CN111182908A - Pharmaceutical compounds and methods for their purification - Google Patents

Abstract

The present invention provides a method of preparing a lyophilized pharmaceutical composition comprising a compound described herein or a pharmaceutically acceptable salt thereof. The method comprises dissolving the compound in a solvent comprising dimethyl sulfoxide and optionally one or more co-solvents to form a solution, and then removing the solvent and any co-solvents by a freeze-drying process. The invention also provides lyophilized pharmaceutical compositions and their use in medicine, in particular in the treatment of cancer.

Description

Pharmaceutical compounds and methods for their purification

Cross-referencing

This application claims the benefit of U.S. provisional application No. 62/540,706, filed on 3/8/2017, which is incorporated herein by reference in its entirety.

Background

DNA methylation is the post-replicative chemical modification of DNA. Different cancers can be stratified by their aberrant DNA methylation profiles (overall or degree of specific DNA methylation), and hypermethylation of specific genes may be associated with prognosis in gastric, lung, esophageal, pancreatic and colon cancers. DNA methylation patterns can also be used to predict glioma and melanoma responses or resistance to treatment. Azacitidine and decitabine are two FDA-approved methylation-reducing agents (HMAs) that exert their therapeutic effects by inhibiting the level of DNA methylation.

Lyophilization, commonly referred to as freeze-drying, is a dehydration process in which a matrix containing a solvent is frozen and then subjected to vacuum such that the solvent is removed by sublimation, i.e., direct conversion from a solid frozen state to a gaseous state.

Is incorporated by reference

Each of the patents, publications, and non-patent documents cited in this application is incorporated by reference herein in its entirety, as if each were individually incorporated by reference.

Disclosure of Invention

In some embodiments, the present invention provides a composition comprising:

a) a compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein the composition comprises at least 95% of the compound; and

b) a nucleotide-based compound which is not a compound of formula (1).

Drawings

Figure 1 is a graph of dimethyl sulfoxide (DMSO) removal over time as the lyophilization process described herein proceeds. Figure 1 shows DMSO removal curves for four formulations A, B, C and D at different concentrations.

Fig. 2 depicts lyophilization parameters for a subject lyophilization process described herein.

Fig. 3 provides a top view of a lyophilized product of a subject lyophilization process described herein.

Figure 4 provides a superimposed plot of differential scanning calorimetry and thermogravimetric analysis thermograms of a target lyophilization process as described herein.

Fig. 5 depicts lyophilization parameters for a target lyophilization process employing low chamber pressure as described herein.

Fig. 6 provides a top view of a lyophilized product of a target lyophilization process described herein that employs a low chamber pressure.

Figure 7 provides a superimposed plot of differential scanning calorimetry and thermogravimetric analysis thermograms of the target lyophilization process described herein using low chamber pressure.

Fig. 8 depicts lyophilization parameters for a high shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 9 depicts residual gas analyzer results of a high shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 10 provides a top view of a lyophilized product of the high shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 11 provides a superimposed plot of differential scanning calorimetry and thermogravimetric analysis thermograms of a high shelf temperature, high chamber pressure lyophilization process as described herein.

Fig. 12 depicts lyophilization parameters for the low shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 13 depicts residual gas analyzer results of a low shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 14 provides a top view of a lyophilized product of the low shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 15 provides a superimposed plot of differential scanning calorimetry and thermogravimetric analysis thermograms of a low shelf temperature, high chamber pressure lyophilization process as described herein.

Fig. 16 depicts lyophilization parameters for the high shelf temperature, low chamber pressure lyophilization process described herein.

Fig. 17 depicts residual gas analyzer results of the high shelf temperature, low chamber pressure lyophilization process described herein.

Fig. 18 provides a top view of a lyophilized product of the high shelf temperature, low chamber pressure lyophilization process described herein.

Fig. 19 provides a superimposed plot of differential scanning calorimetry and thermogravimetric analysis thermograms of a high shelf temperature, low chamber pressure lyophilization process as described herein.

Fig. 20 depicts lyophilization parameters for a low shelf temperature, low chamber pressure lyophilization process described herein.

Panels a and B in fig. 21 provide top views of the lyophilized products of batch 1 and batch 2, respectively, of the compound of formula (1) used in the low shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 22 provides an overlay of differential scanning calorimetry and thermogravimetric thermograms of the low shelf temperature, low chamber pressure lyophilization process described herein.

Fig. 23 depicts lyophilization parameters for a subject lyophilization process described herein.

Panel a in fig. 24 and panel B in fig. 24 provide top views of the lyophilized products of batch 1 and batch 2, respectively, of the compound of formula (1) for use in the target lyophilization process described herein.

Fig. 25 provides a superimposed plot of differential scanning calorimetry and thermogravimetric analysis thermograms of a target lyophilization process as described herein.

Fig. 26 depicts lyophilization parameters for a low shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 27 provides a top view of a lyophilized product of the low shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 28 provides a superimposed plot of differential scanning calorimetry and thermogravimetric analysis thermograms of a low shelf temperature, high chamber pressure lyophilization process as described herein.

Fig. 29 depicts lyophilization parameters for a high shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 30 depicts residual gas analyzer results of a high shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 31 provides a top view of a lyophilized product of the high shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 32 provides a superimposed plot of differential scanning calorimetry and thermogravimetric analysis thermograms of a high shelf temperature, high chamber pressure lyophilization process as described herein.

Fig. 33 depicts the lyophilization parameters for a repeat study of the high shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 34 provides a side view of a lyophilized product of a repeat study of the high shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 35 provides a top view of a lyophilized product of a repeat study of the high shelf temperature, high chamber pressure lyophilization process described herein.

Fig. 36 provides an overlay of differential scanning calorimetry and thermogravimetric analysis thermograms of a repeat study of the high shelf temperature, high chamber pressure lyophilization process described herein.

Figure 37 provides the lyophilization cycle parameter results of table 6.

Figure 38 provides RGA data for the lyophilization parameters of table 6.

Fig. 39 shows a top view of the vial of lyophilized product of table 6.

Figure 40 provides the thermogravimetric thermograms of the studies of table 6.

Detailed Description

The present application relates to lyophilized pharmaceutical compositions containing dinucleotides derived from decitabine, and to methods of making and using dinucleotides compositions derived from decitabine.

The present disclosure relates to improved lyophilized compositions containing a compound of formula (1) or a pharmaceutically acceptable salt thereof, and to methods of making the improved lyophilized pharmaceutical compositions using a lyophilization process. The present disclosure also provides for the use of the lyophilized pharmaceutical composition in medicine, in particular the use of the lyophilized pharmaceutical composition in the treatment of cancer.

The present disclosure provides methods for lyophilizing a matrix comprising a non-aqueous solvent, such as DMSO, and a compound of formula (1), or a pharmaceutically acceptable salt thereof. Typically, the method comprises two freezing stages, with an intermediate warming stage (annealing stage) between the two freezing stages. The method may be used to remove non-aqueous solvents from a substrate. In some embodiments, the compound within the matrix is a compound of formula (1):

or a pharmaceutically acceptable salt thereof. The present disclosure also provides lyophilized compositions comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof. In addition, the present disclosure provides the use of the lyophilized pharmaceutical composition in medicine, in particular in the treatment of cancer.

By using two freezing stages and an intermediate warming stage (annealing stage) in between, DMSO can be removed more quickly during the subsequent primary drying stage and, therefore, the length of the secondary drying stage can be greatly shortened. This intermediate warming stage may provide increased porosity, making DMSO more sublimable. Thus, more DMSO may be removed during the primary drying stage.

Freeze-drying microscopy (FDM) studies of the formulations have shown that some residual non-freezing solvent or co-solvent may sometimes be present even at temperatures below-30 ℃. Thus, the term "frozen" as used herein includes such states: where there is a solid structure formed by solvent and/or co-solvent molecules, but there may also be some solvent and/or co-solvent in non-frozen or liquid form.

Method for preparing freeze-dried pharmaceutical composition

The methods provided herein include a method of preparing a lyophilized pharmaceutical composition comprising a compound (e.g., a compound of formula (1)) or a pharmaceutically acceptable salt thereof, the method comprising dissolving a compound of formula (1) or a pharmaceutically acceptable salt thereof in a non-aqueous solvent that may comprise DMSO and optionally one or more co-solvents to form a solution, and then removing the solvent and any co-solvents by a lyophilization process to obtain a lyophilized product; wherein the freeze-drying process may comprise one or more of the following stages: (i) a first freezing stage in which the solution is frozen by reducing its temperature to a temperature of no more than-20 ℃; (ii) a first warming phase in which the temperature of the frozen solution is raised to a temperature in the range-15 ℃ to 5 ℃, at which temperature the solution remains in a frozen state; (iii) a second freezing phase which occurs after the first warming phase and in which the temperature of the solution in the frozen state is reduced to a temperature of not higher than-20 ℃; (iv) a primary drying stage comprising a sublimation step, wherein DMSO and the co-solvent(s) (if present) are removed by sublimation from the solution in the frozen state under reduced pressure to give a partially dried product; and (v) a secondary drying stage in which the DMSO and the co-solvent(s), if present, are removed by evaporation under reduced pressure from the partially dried product in a non-frozen state to give a lyophilised product.

The sequence of freezing and intermediate warming stages (i), (ii) and (iii) may be repeated one or more times before the primary drying stage (iv) is carried out. For example, the first sequence of stages (i), (ii) and (iii) may be followed by the second sequence of stages (i), (ii) and (iii), and optionally followed by the third and fourth sequence of stages (i), (ii) and (iii), followed by the primary drying stage (iv).

For example, the methods described herein can shorten the total time of the freeze-drying process by at least one day, and in some embodiments, by up to two days. The methods described herein may further allow for easier reconstitution of the solution as compared to compositions prepared using methods that omit an intermediate warming stage. For example, in some embodiments, the reconstitution time of the composition may be reduced from a time of more than 30 minutes to a time of less than 20 minutes, and in some embodiments, to a time of less than 10 minutes.

The freeze-drying process may be performed in a freeze-drying apparatus. The lyophilization apparatus may have a chamber in which a lyophilization container (e.g., a lyophilization vial) containing a solution may be placed for lyophilization. The chamber may be connected to a vacuum source (e.g., a vacuum pump) to enable the pressure within the chamber to be reduced. The apparatus may also have an assembly for freezing or heating the contents of the chamber. Prior to lyophilization, a bulk solution of the compound of formula (1) in DMSO and optionally one or more co-solvents can be prepared and filtered through a filter (e.g., a sterile filter), after which aliquots are filled into lyophilization containers (e.g., lyophilization vials) and transferred to a lyophilization apparatus. The container may be partially stoppered to prevent contamination prior to transfer to the lyophilization apparatus, but still allow the solvent to escape during the lyophilization process.

For each parameter provided herein, the freeze-drying process parameters are set forth in more detail with reference to particular embodiments, groups, subgroups, ranges, and individual values. Each embodiment, set, subgroup, range and individual value defined for one parameter of the freeze-drying process may be combined with each embodiment, set, subgroup, range and individual value defined for any other parameter of the freeze-drying process. Accordingly, the present application discloses all combinations of embodiments, groups, subgroups, ranges and individual values for each parameter of the freeze-drying process.

The temperatures mentioned above and elsewhere herein in relation to the parameters of the lyophilization process are the temperatures of the shelves in the lyophilization apparatus. The shelves may be cooled by a cooling fluid, the temperature of which is monitored, and a method of determining the temperature of the shelves is provided. The temperature measurements obtained from the cooling fluid can be cross-checked with the temperature obtained directly from the product in the lyophilization vessel by inserting a temperature probe into the selected lyophilization vessel.

In the first freezing stage (i), the solution may be frozen by reducing its temperature to a temperature of not higher than about-20 ℃, for example, the temperature may be reduced to a value of not higher than about-30 ℃ (or not higher than about-35 ℃, or not higher than about-40 ℃, or not higher than about-41 ℃, or not higher than about-42 ℃, or not higher than about-43 ℃, or not higher than about-44 ℃). For example, the solution may be frozen by reducing the temperature to a value of about-40 ℃ to about-50 ℃, or about-42 ℃ to about-48 ℃, or about-43 ℃ to about-47 ℃, or about-44 ℃ to about-46 ℃, or about-45 ℃.

The first freezing stage may comprise a temperature ramping step in which the temperature is reduced from an initial (e.g. ambient) temperature to a target temperature over a first period of time, for example over a period of up to about 2 hours or up to about 1.5 hours or up to 1.25 hours, or up to about 1 hour.

Once the target temperature is reached, the frozen solution may be held at the target temperature for a second period of time, such as up to about 3 hours, or up to about 2.5 hours, or up to about 2 hours, or up to about 1.5 hours.

After the first freezing stage, the solution may be subjected to a first warming stage in which the temperature of the frozen solution is raised to a temperature in the range of-15 ℃ to 4 ℃, at which temperature the solution remains frozen. For example, the frozen solution can be warmed to a temperature of about-5 ℃ to about 5 ℃, or about-3 ℃ to about 3 ℃, or about-2 ℃ to about 2 ℃, or about-1 ℃ to about 1 ℃, e.g., about 0 ℃.

The first warming phase may include a first period of time during which the frozen solution is warmed to a target temperature and a second period of time during which the frozen solution is maintained at the target temperature. For example, the first period of time to warm the frozen solution to the target temperature may be up to about 2 hours, or up to about 1.75 hours, or up to about 1.5 hours, or up to about 1.3 hours, or up to about 1.2 hours, or up to about 1.1 hours, or up to about 1 hour.

After the first warming phase, the still frozen solution may be subjected to a second freezing phase in which the temperature of the solution in the frozen state is reduced to a temperature of no greater than about-20 ℃. The temperature may be reduced to a value of no greater than about-30 deg.C (or no greater than about-35 deg.C, or no greater than about-40 deg.C, or no greater than about-41 deg.C, or no greater than about-42 deg.C, or no greater than about-43 deg.C, or no greater than about 44 deg.C). For example, the temperature of the frozen solution can be reduced to a value of about-40 ℃ to about-50 ℃, or about-42 ℃ to about-48 ℃, or about-43 ℃ to about-47 ℃, or about-44 ℃ to about-46 ℃, such as about-45 ℃.

After the second freezing stage, the frozen solution may be subjected to a primary drying stage comprising a sublimation step wherein dimethyl sulfoxide and the co-solvent(s), if present, are removed by sublimation from the solution in the frozen state under reduced pressure to give a partially dried product. During the primary drying stage, the frozen solution may be warmed to promote faster sublimation of DMSO while maintaining the solution in a frozen state. For example, the frozen solution may be warmed to a temperature in the range of-25 ℃ to 0 ℃, or-22 ℃ to-2 ℃, e.g., about-20 ℃ to about-5 ℃.

In the primary drying stage, the frozen solution may be warmed in steps. For example, in a first warming step, the temperature may be raised from a temperature of no greater than about-30 ℃ to a temperature in the range of about-25 ℃ to about-19 ℃ (e.g., about-20 ℃) and then held at that temperature for a defined holding period. At this temperature, residual unfrozen solvent and/or co-solvent may be removed by evaporation.

In the second temperature raising step, the temperature may be raised from a temperature in the range of about-25 ℃ to about-19 ℃ (e.g., about-20 ℃) to a temperature in the range of about-10 ℃ to about 0 ℃ (e.g., about-5 ℃), and then held at that temperature for a further defined holding time. Further intermediate ramp-up and hold periods may be added to the first and second ramp-up stages. As an alternative to a staged warming of the frozen solution, the warming may be performed in a continuous manner until the desired target temperature is reached.

At the beginning of the primary drying period, the pressure in the vessel containing the frozen solution may be reduced (typically from atmospheric pressure) to a pressure at which the DMSO and optional other co-solvent may be removed. The pressure can be reduced to a pressure below 1mBar, for example below 500 μ Bar, or below 100 μ Bar, or below 50 μ Bar. For example, the pressure may be reduced to a pressure below 20 μ Bar, or below 10 μ Bar, or 1 to 10 μ Bar, or 4 to 8 μ Bar, for example about 6 μ Bar.

The primary drying stage may include an initial depressurization stage in which the temperature is held constant and the pressure is reduced to a target value, followed by warming of the frozen solution as described above. Alternatively, the pressure reduction and the temperature increase of the frozen solution may be performed simultaneously.

The primary drying stage may take from about 20 to about 60 hours, for example from about 30 to about 50 hours.

The progress of the primary drying stage can be monitored by one or more sensors or meters present in the lyophilization chamber of the lyophilization apparatus. A sensor or meter, such as a Pirani vacuum gauge (Pirani gauge), may be used to measure one or more parameters within the chamber, whereby a determined change in the one or more parameters may indicate the progress of primary drying and provide a means to determine when sublimation of the DMSO and optionally any co-solvent is complete. For example, a sensor or meter may measure the pressure within the chamber or the conductance of a gas within the chamber.

During sublimation, the temperature must be below the critical temperature and pressure of the product to keep the product frozen. Sublimation is a direct solid to gas DMSO phase change. If the conditions are above the critical temperature and pressure, the product does not freeze but is liquid and the DMSO can change from liquid to gas (boil).

The primary drying stage may be carried out at a pressure of about 5 μ Bar to about 40 μ Bar. The freezing temperature of the product at these pressures is from about-2 ℃ to about-4 ℃. The primary drying stage may be carried out at a temperature of from about-3 ℃ to about-9 ℃. In this temperature range, the vapor pressure is sufficient for rapid sublimation, which results in better products. In some embodiments, the pressure is about 20 μ Bar. In some embodiments, the temperature is about-6 ℃.

Once the sublimation of DMSO has stopped, or has fallen below a certain level, the secondary drying phase is started. In the secondary drying stage, the dimethyl sulfoxide and, if present, the co-solvent(s) are removed by evaporation under reduced pressure from the partially dried product in the non-frozen state to give a lyophilized product. Thus, in the secondary drying stage, a reduced pressure environment is maintained and the partially dried product is heated to a temperature at which the product no longer freezes. Since DMSO has a boiling point of about 189 ℃, the partially dried product can be heated to a temperature of at least about 40 ℃, more typically at least about 45 ℃, e.g., at least about 50 ℃, or at least about 55 ℃. In some embodiments, the partially dried product is heated to a temperature in the range of from about 55 ℃ to about 70 ℃, for example, about 65 ℃.

The secondary drying stage may comprise one or more temperature ramping steps in which the partially dried product is heated to a target temperature, each temperature ramping step being followed by a temperature maintenance step. In one embodiment, there is a single temperature ramp step followed by a single temperature hold step.

In a secondary drying stage, unfrozen solvent molecules are removed to give a lyophilized product containing only small amounts of residual DMSO.

The secondary drying stage may be carried out at a temperature of from about 30 ℃ to about 65 ℃, for example about 40 ℃.

At the end of the secondary drying phase, an inert gas, such as nitrogen, is introduced into the lyophilization chamber, and the container (e.g., vial) containing the lyophilized product is completely sealed under the inert gas (e.g., by means of a stopper and optionally a lid).

A solution of a compound of formula (1) or a pharmaceutically acceptable salt thereof in a non-aqueous solvent comprising dimethyl sulfoxide and optionally one or more co-solvents may be subjected to a freeze-drying procedure.

A break temperature as described herein may refer to a pause step in the lyophilization process for which a target shelf temperature has not been specified. At the interrupt temperature, the temperature in the chamber begins to rise when the DMSO sublimation during lyophilization is complete. The interrupt temperature may be indicated by an elevated product temperature measured after the primary drying step when the process reaches steady state.

In some embodiments, water contamination is avoided at any stage. The formation of hydrates can destroy the structure of the product, making it difficult to reconstitute easily.

In some embodiments, a co-solvent is substantially absent; i.e. the solvent consists essentially of DMSO.

In other embodiments, one or more other non-aqueous co-solvents may be present. Where a co-solvent is present, the total volume of co-solvent may generally comprise no more than about 25% (v/v) of the total solvent. More typically, when present, the total volume of co-solvent comprises no more than about 20%, or no more than about 15%, or no more than about 10%, or no more than about 5% (by volume) of the total volume of solvent. For example, the total volume of co-solvent may be from about 0% (v/v) to about 5% (v/v) of the total volume of solvent.

The solution to be lyophilized may contain the compound of formula (1) or a pharmaceutically acceptable salt thereof in an amount in the range of about 5mg/ml to about 200mg/ml, for example in the range of about 10mg/ml to about 150 mg/ml. For example, the solution may contain from about 20mg/ml to about 120mg/ml or from about 22mg/ml to about 110mg/ml or from about 25mg/ml to about 105mg/ml or from about 25mg/ml to about 100mg/ml of a compound of formula (1) or a pharmaceutically acceptable salt thereof.

In some embodiments, the solution contains from about 40mg/ml to about 110mg/ml or from about 50mg/ml to about 105mg/ml of the compound of formula (1) or a pharmaceutically acceptable salt thereof.

In some embodiments, the solution contains 75mg/ml or 100mg/ml of the sodium salt of the compound of formula (1).

Non-limiting examples of pressures that can be used in the methods described herein include about 1 μ Bar, about 2 μ Bar, about 3 μ Bar, about 4 μ Bar, about 5 μ Bar, about 6 μ Bar, about 7 μ Bar, about 8 μ Bar, about 9 μ Bar, about 10 μ Bar, about 15 μ Bar, about 20 μ Bar, about 25 μ Bar, about 30 μ Bar, about 35 μ Bar, about 40 μ Bar, about 45 μ Bar, about 50 μ Bar, about 55 μ Bar, about 60 μ Bar, about 65 μ Bar, about 70 μ Bar, about 80 μ Bar, about 90 μ Bar, about 100 μ Bar, about 150 μ Bar, about 200 μ Bar, about 250 μ Bar, about 300 μ Bar, about 350 μ Bar, about 400 μ Bar, about 450 μ Bar, about 500 μ Bar, about 550 μ Bar, about 650 μ Bar, about 800 μ Bar.

Non-limiting examples of pressures that may be used in the methods described herein include about 0PSI, about 0.1PSI, about 0.15PSI, about 0.2PSI, about 0.25PSI, about 0.3PSI, about 0.35PSI, about 0.4PSI, about 0.45PSI, about 0.5PSI, about 0.55PSI, about 0.6PSI, about 0.65PSI, about 0.7PSI, about 0.75PSI, about 0.8PSI, about 0.85PSI, about 0.9PSI, about 0.95PSI, about 1PSI, about 1.1PSI, about 1.2PSI, about 1.3PSI, about 1.4PSI, about 1.5PSI, about 1.6PSI, about 1.7PSI, about 1.8PSIG, about 1.9PSI, about 2PSI, about 2.1PSI, about 2.2PSI, about 2.3, about 2.4PSI, about 2.5PSI, about 2.7PSI, about 2.8PSI, about 3, about 2.9PSI, about 2.8PSI, about 3, about 2.8PSI, about 2.9PSI, about 2.8 PSI.

Non-limiting examples of pressures that may be used in the methods described herein include about 0.5PSIG (PSIG), about 0.6PSIG, about 0.7PSIG, about 0.8PSIG, about 0.9PSIG, about 1PSIG, about 1.1PSIG, about 1.2PSIG, about 1.3PSIG, about 1.4PSIG, about 1.5PSIG, about 1.6PSIG, about 1.7PSIG, about 1.8PSIG, about 1.9PSIG, about 2PSIG, about 2.5PSIG, about 3PSIG, about 3.5PSIG, about 4PSIG, about 4.5PSIG, about 5PSIG, about 6PSIG, about 7PSIG, about 8PSIG, about 9PSIG, about 10PSIG, about 15PSIG, about 20PSIG, about 25PSIG, about 30PSIG, about 35PSIG, about 40PSIG, about 45PSIG, or about 55 PSIG.

Non-limiting examples of pressures that may be used in the processes described herein include about 5PSIA (absolute PSI), about 6PSIA, about 7PSIA, about 8PSIA, about 9PSIA, about 10PSIA, about 10.5PSIA, about 11PSIA, about 11.5PSIA, about 12PSIA, about 12.5PSIA, about 13PSIA, about 13.5PSIA, about 14PSIA, about 14.1PSIA, about 14.2PSIA, about 14.3PSIA, about 14.4PSIA, about 14.5PSIA, about 14.6PSIA, about 14.7PSIA, about 14.8PSIA, about 14.9PSIA, about 15 PSIA, about 16PSIA, about 17PSIA, about 18PSIA, about 19PSIA, or about 20 PSIA.

Non-limiting examples of pressures that may be used in the methods described herein include about 1 micron (mTorr), about 2 microns, about 3 microns, about 4 microns, about 5 microns, about 6 microns, about 7 microns, about 8 microns, about 9 microns, about 10 microns, about 15 microns, about 20 microns, about 25 microns, about 30 microns, about 35 microns, about 40 microns, about 45 microns, about 50 microns, about 55 microns, about 60 microns, about 65 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, about 150 microns, about 200 microns, about 250 microns, about 300 microns, about 350 microns, about 400 microns, about 450 microns, about 500 microns, about 550 microns, about 600 microns, about 650 microns, about 700 microns, about 750 microns, about 800 microns, about 850 microns, about 900 microns, about 950 microns, and about 1000 microns.

Solution reconstitution: the requirement of the reconstituted solution is that after a predetermined length of time, no insoluble material is visible and the clarity of the solution is no inferior to that of the diluent. The volume used for reconstitution may return the product to the same volume and concentration as the bulk solution used for filling, or may be the volume intended for patient delivery in a clinical setting.

For reconstitution, a specified volume of diluent may be drawn into the syringe. The diluent can then be extruded into the center of the product cake and the timer started. The product was then examined at approximately 5 second intervals to determine when the material dissolved.

The method used herein to evaluate the lyophilization process.

Hastings Meter (thermocouple Meter): thermocouple-type vacuum gauges are instruments that measure pressure indirectly based on heat conduction through a gas. The pressure of the vessel can be measured by the temperature fluctuations of the "hot filament" caused by the collision of gas molecules with the filament. When the pressure is in the low vacuum range (e.g., > 100 microns), the number of gas molecules that collide with the hot filament is high. Since each gas molecule absorbs a certain amount of heat upon collision with the wire, there is a greater cooling effect on the wire, thereby reducing the relative temperature measured by the thermocouple. With the wire held at a constant voltage, the temperature change can be correlated to the relative vacuum level that can be indicated on the instrument.

When the Hastings meter monitors the pressure within a chamber of a lyophilizer such as that used herein, the meter can be used as an indicator of the gaseous environment within the chamber. The Hastings instrument is adjusted in a pure nitrogen environment; thus, the meter reads the chamber pressure based on the thermal conductivity of the nitrogen. When the chamber environment contains solvent vapor from the product during primary and secondary drying, the Hastings gauge reads an artificially high pressure due to the difference in thermal conductivity of the solvent vapor compared to nitrogen. This deviation can be measured by comparing the Hastings meter reading with the capacitance manometer reading (which is used to control the chamber pressure).

When the solvent vapor level in the chamber environment drops, the readings of the Hastings meter are returned to match the readings of the capacitance manometer, indicating the end of sublimation in primary drying or the end of desorption in secondary drying. The sensitivity of the Hastings instrument depends on the variation in thermal conductivity, which can depend on the relative difference in thermal conductivity of the solvent vapor and nitrogen in the chamber environment and the ratio of solvent vapor to nitrogen.

Residual Gas Analyzer (RGA): RGA is a mass spectrometer that can monitor the chamber environment at sub-atmospheric pressures using quadrupole mass analyzer technology. MKS Microvision Plus RGA was connected to a port on the chamber. Using ProcessEye^TMAnd the Professional software realizes test parameters and data collection. RGA may be at 2x 10^-4To 2x 10^-9The pressure range of Torr resolves the components with atomic mass of 1 to 90 in the environment.

RGAs can be programmed to scan the chamber every 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or one hour. In some embodiments, the RGA scans the chamber every 5 minutes.

The automatic valve isolates the RGA from the chamber until the pressure in the chamber is below 1000 microns. The orifices may be lined up between the chamber and the RGA to induce the pressure drop necessary to maintain the instrument pressure within the desired range. During primary drying, the relative amounts of DMSO and nitrogen present in the chamber were monitored to confirm the end of sublimation of the ice. The NIST of DMSO reports a major ion of 63 Atomic Mass Units (AMU), the parent compound is 78AMU, and a minor peak.

Turbidity: turbidity the transmission of light through a liquid sample is monitored to determine whether the sample is clear or to determine the opacity of the sample. The analysis can be performed using a Hach Model 2100AN laboratory turbidimeter. Hach Model 2100AN is a ratiometric turbidimeter that uses the ratio of transmitted light to scattered light to reduce measurement errors caused by colored solutions.

Before each use, Hach was used

The turbidimeter was calibrated using a calibration kit, and then Hach was used

And (5) checking the second-level turbidity standard. The sample cell is cleaned and oiled to reduce interference caused by dirt or imperfections on the glass. Sample cell indexing can match sample cells with similar interference for sample comparison.

Reconstituted samples can be combined to achieve at least 2.5mL of sample, which is placed in an indexed sample cell for analysis. The turbidimeter measures the opacity and returns the value as the turbidity unit (NTU) ratio of the turbidity measurement. This value is recorded by the instrument with a time and date stamp to track each sample.

High temperature differential scanning calorimetry (HT-DSC): high temperature modulated differential scanning calorimetry (MDSC, 2 ℃/min) was used as a means to determine the thermal properties of solid materials. HT-DSC follows current USP<891>Thermal analysis was performed using a TAInstructions Q200. Using TA Instruments Universal

Software version 4.5A performs test parameters and data analysis.

Briefly, solid material weighing 3mg to 6mg was placed in an aluminum sample pan with a crimped vent cover. The sample was continuously purged with nitrogen NF at a flow rate of 50 mL/min. The sample was heated from 20 ℃ to 200 ℃ at a rate of 10 ℃/min or 2 ℃/min (0.32 ℃ C. every 60 seconds). The instrument was calibrated at temperatures that span the range of the pyrometry performed herein.

Physical inspection: physical inspection can be used to assess the uniformity of appearance of the end product, for example in terms of color, texture, shape and structure, and can provide insight into the relative effectiveness of the treatment on the finished sample. The degree, extent, and/or consistency of each attribute may be considered and recorded.

A color may be characterized as the intensity, chroma, or hue of the color, indicating the hue and shade reflecting the lightness to darkness of the color. The product structure may be described as dense or open, granular or geometric-like in shape, or as a composition of an arrangement, configuration, pattern or organization that makes up the structure. Texture can be characterized as smooth to fine, looking like powdered sugar or chalk with limited structures that are difficult to distinguish, and rough texture where structures are easily observed.

Each sample can be viewed at the bottom, side and top of the cake while rotating the container to view all sides.

Thermogravimetric analysis (TGA): TGA can be used as a confirmatory method for residual moisture determination, where changes in weight are attributed to the evolution of volatile species such as water. In addition, TGA can be used to determine physicochemical changes as the sample begins to decompose at elevated temperatures.

TGA monitors the change in weight of the material with heating temperature or time. The analysis may be performed using a TAInstructions QSO according to the thermal analysis of USP 891. TA Instruments Universal can be used on the PC interface

Software version 4.5A performs test parameters and data analysis.

Solid material weighing about 13mg to about 19mg may be placed in an open ceramic sample pan. The sample was then heated from 25 ℃ to 400 ℃ at a ramp rate of about 10 ℃ per minute to measure weight loss over the entire temperature range. The sample was continuously purged with nitrogen NF at a flow rate of 60 mL/min. The instrument was calibrated at temperatures spanning the range of pyrometry.

The lyophilized material is warmed and any change in sample weight can be monitored. During the temperature rise, the weight loss is associated with the evolution of volatile components in the sample. The sample weight related to the temperature was determined by calculation.

Lyophilized pharmaceutical composition

The present disclosure provides lyophilized pharmaceutical compositions that can be prepared (or have been prepared) by the lyophilization process described herein.

The lyophilized pharmaceutical compositions of the present disclosure are characterized by enhanced solubility relative to known lyophilized formulations of the compound of formula (1) and salts thereof. Thus, in another embodiment, the present disclosure provides a lyophilized pharmaceutical composition comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, obtainable by a lyophilization process as defined herein, and having a dissolution time of no more than 20 minutes in a non-aqueous solvent comprising 65% (v/v) propylene glycol, 25% (v/v) glycerol and 10% (v/v) ethanol at ambient temperature and without the aid of mechanical stirring.

In some embodiments, the dissolution time of the lyophilized pharmaceutical composition in a non-aqueous solvent is no more than 15 minutes, or no more than 12 minutes.

In a particular embodiment, the dissolution time of the lyophilized pharmaceutical composition in a non-aqueous solvent is no more than 10 minutes.

The lyophilized pharmaceutical compositions described herein are also characterized by a reduced level of residual DMSO solvent. Thus, in another embodiment, the present disclosure provides a lyophilized pharmaceutical composition comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, which is obtainable by a lyophilization process as defined herein, and wherein in an amount of the lyophilized composition obtained from 1 gram of solution, there is a residual DMSO content of no more than 20mg or no more than 19 mg. The solution may be a solution of a pharmaceutically acceptable salt thereof in a solvent comprising dimethyl sulfoxide and optionally one or more co-solvents. The solvent may be non-aqueous, anhydrous or substantially anhydrous.

In another embodiment, there is provided a lyophilized pharmaceutical composition comprising a compound of formula (1), or a pharmaceutically acceptable salt thereof, which is obtainable by a lyophilization process as defined herein, and wherein any residual DMSO is present in the composition in an amount equivalent to no more than 35mg/100mg equivalent of the free base of the compound of formula (1).

The term "100 mg equivalents of the free base" may refer to the amount by weight of the free base which may be present, or when the compound of formula (1) is in the form of a salt, the amount by weight of the free base contained within the salt. For example, the amount of residual DMSO does not exceed about 32mg or does not exceed about 31mg per 100mg equivalent of free base, such as in the range of about 15mg to about 35mg, or about 20mg to about 32mg, or about 25mg to about 30 mg.

In some embodiments, there is provided a lyophilized pharmaceutical composition comprising a compound of formula (1), or a pharmaceutically acceptable salt thereof, which is obtainable by a freeze-drying process as defined herein, and which: (a) a dissolution time in a solvent comprising 65% (v/v) propylene glycol, 25% (v/v) glycerol and 10% (v/v) ethanol of no more than 20 minutes (or no more than 15 or 12 or 10 minutes) at ambient temperature and without mechanical agitation; and (b) has a residual DMSO content such that in an amount of the lyophilized composition obtained from 1 gram of the solution, the residual DMSO content does not exceed 20mg or does not exceed 19 mg. The solvent may be non-aqueous, anhydrous or substantially anhydrous.

The lyophilized pharmaceutical compositions described herein, i.e., compositions obtainable by the lyophilization process described herein, can also be characterized in terms of their increased porosity and increased specific surface area as compared to known compositions. The specific surface area can be measured using known techniques such as the Brunauer-Emmett-Teller (BET) adsorption method.

The lyophilized pharmaceutical compositions described herein can be provided in a sealed container, such as a vial (e.g., a glass vial), optionally containing a protective atmosphere of an inert gas, such as nitrogen or argon. The sealed container can be opened when needed and its contents reconstituted by dissolution in a reconstitution solvent, such as a non-aqueous, anhydrous or substantially anhydrous solvent, prior to administration to a patient.

The present disclosure further provides a sealed pharmaceutical container containing a lyophilized pharmaceutical composition as described herein. The sealed medicament container may be, for example, a vial containing a stopper and optionally an additional component (e.g., a collar) for holding the stopper in place. The sealed vessel may optionally contain a protective atmosphere of an inert gas such as nitrogen or argon.

In some embodiments, the present disclosure provides a sealed pharmaceutical container containing a lyophilized pharmaceutical composition as described herein, wherein the composition contains a compound of formula (1), or a pharmaceutically acceptable salt thereof, in an amount equivalent to about 100mg equivalent of free base of the compound of formula (1), and wherein no more than 35mg of residual DMSO is present in the composition.

Reconstituted formulations prepared from lyophilized pharmaceutical compositions

The lyophilized pharmaceutical compositions described herein can be reconstituted in a solvent, such as a non-aqueous, anhydrous, or substantially anhydrous solvent, to yield an injectable liquid composition for administration to a subject. The liquid composition may be for administration by subcutaneous injection. The present disclosure further provides a method of preparing an injectable liquid composition, which method may comprise dissolving a lyophilized pharmaceutical composition as described herein in a solvent, particularly a non-aqueous solvent.

Non-limiting examples of suitable solvents include propylene glycol, glycerol, ethanol, and any combination of the foregoing. The preparation can be prepared into non-aqueous preparation. The formulation may be anhydrous or substantially anhydrous.

The mixture of solvents may contain a percentage of propylene glycol on a mass or volume basis. In some embodiments, the percentage of propylene glycol may be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In some embodiments, the percentage of propylene glycol may be up to 90%, up to 80%, up to 70%, up to 60%, up to about 90%, up to about 80%, up to about 70%, or up to about 60%. In some embodiments, the percentage of propylene glycol may be from about 30% to about 90%, from about 45% to about 85%, from about 55% to about 75%, from about 60% to about 70%, from about 30% to about 90%, from about 45% to about 85%, from about 55% to about 75%, or from about 60% to about 70%. In some embodiments, the percentage of propylene glycol may be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%.

The mixture of solvents may contain a percentage of glycerol on a mass or volume basis. In some embodiments, the percentage of glycerol may be at least 5%, at least 10%, at least 15%, at least 25%, at least 30%, at least about 5%, at least about 10%, at least about 15%, at least about 25%, or at least about 30%. In some embodiments, the percentage of glycerol may be at most 70%, at most 60%, at most 5%, at most 40%, at most 30%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, or at most about 30%. In some embodiments, the percentage of glycerol may be 0% to 50%, 5% to 45%, 15% to 35%, 20% to 30%, 0% to about 50%, about 5% to about 45%, about 15% to about 35%, or about 20% to about 30%. In some embodiments, the percentage of glycerol may be 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

The mixture of solvents may contain a percentage of ethanol on a mass or volume basis. In some embodiments, the percentage of ethanol may be at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least about 1%, at least about 3%, at least about 5%, at least about 10%, or at least about 15%. In some embodiments, the percentage of ethanol may be at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, or at most about 10%. In some embodiments, the percentage of ethanol may be 0% to 30%, 0% to 25%, 0% to 20%, 5% to 15%, 0% to about 30%, 0% to about 25%, 0% to about 20%, or about 5% to about 15%. In some embodiments, the percentage of ethanol may be 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%.

In some embodiments, the solvent or solvent mixture contains 45% to 85% propylene glycol, 5% to 45% glycerol, and 0% to 30% ethanol. In some embodiments, the solvent or solvent mixture contains about 45% to about 85% propylene glycol, about 5% to about 45% glycerol, and 0% to about 30% ethanol. In some embodiments, the solvent or solvent mixture consists essentially of 45% to 85% propylene glycol, 5% to 45% glycerol, and 0% to 30% ethanol. In some embodiments, the solvent or solvent mixture consists essentially of about 45% to about 85% propylene glycol, about 5% to about 45% glycerin, and 0% to about 30% ethanol. In some embodiments, the solvent or solvent mixture is 45% to 85% propylene glycol, 5% to 45% glycerol, and 0% to 30% ethanol. In some embodiments, the solvent or solvent mixture is about 45% to about 85% propylene glycol, about 5% to about 45% glycerol, and 0% to about 30% ethanol.

In some embodiments, the solvent or solvent mixture comprises 55% to 75% propylene glycol, 15% to 35% glycerol, and 0% to 20% ethanol. In some embodiments, the solvent or solvent mixture comprises from about 55% to about 75% propylene glycol, from about 15% to about 35% glycerol, and from 0% to about 20% ethanol. In some embodiments, the solvent or solvent mixture consists essentially of 55% to 75% propylene glycol, 15% to 35% glycerol, and 0% to 20% ethanol. In some embodiments, the solvent or solvent mixture consists essentially of about 55% to about 75% propylene glycol, about 15% to about 35% glycerin, and 0% to about 20% ethanol. In some embodiments, the solvent or solvent mixture is 55% to 75% propylene glycol, 15% to 35% glycerol, and 0% to 20% ethanol. In some embodiments, the solvent or solvent mixture is about 55% to about 75% propylene glycol, about 15% to about 35% glycerol, and 0% to about 20% ethanol.

In some embodiments, the solvent or solvent mixture comprises 60% to 70% propylene glycol, 20% to 30% glycerol, and 5% to 15% ethanol. In some embodiments, the solvent or solvent mixture comprises from about 60% to about 70% propylene glycol, from about 20% to about 30% glycerol, and from about 5% to about 15% ethanol. In some embodiments, the solvent or solvent mixture consists essentially of 60% to 70% propylene glycol, 20% to 30% glycerol, and 5% to 15% ethanol. In some embodiments, the solvent or solvent mixture consists essentially of about 60% to about 70% propylene glycol, about 20% to about 30% glycerin, and about 5% to about 15% ethanol. In some embodiments, the solvent or solvent mixture is 60% to 70% propylene glycol, 20% to 30% glycerol, and 5% to 15% ethanol. In some embodiments, the solvent or solvent mixture is about 60% to about 70% propylene glycol, about 20% to about 30% glycerol, and about 5% to about 15% ethanol.

In some embodiments, the solvent or solvent mixture comprises 65% propylene glycol, 25% glycerol, and 10% ethanol. In some embodiments, the solvent or solvent mixture comprises about 65% propylene glycol, about 25% glycerol, and about 10% ethanol. In some embodiments, the solvent or solvent mixture consists essentially of 65% propylene glycol, 25% glycerol, and 10% ethanol. In some embodiments, the solvent or solvent mixture consists essentially of about 65% propylene glycol, about 25% glycerol, and about 10% ethanol. In some embodiments, the solvent or solvent mixture is 65% propylene glycol, 25% glycerol, and 10% ethanol. In some embodiments, the solvent or solvent mixture is about 65% propylene glycol, about 25% glycerol, and about 10% ethanol.

Excipient

The pharmaceutical compositions described herein may be a combination of any of the pharmaceutical compounds described herein with other chemical components such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. The pharmaceutical compositions can be administered in therapeutically effective amounts as pharmaceutical compositions by a variety of forms and routes including, for example, intravenous, subcutaneous, intramuscular, oral, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, otic, nasal and topical administration.

The pharmaceutical compositions may be administered in a local or systemic manner, for example, by direct injection of the compound into the organ, optionally in the form of a depot or sustained release formulation. The pharmaceutical composition may be provided in the form of a rapid release formulation, in the form of an extended release formulation or in the form of an intermediate release formulation. The quick release form may provide immediate release. Extended release formulations may provide controlled release or sustained delayed release.

For oral administration, pharmaceutical compositions can be readily formulated by combining the active compound with a pharmaceutically acceptable carrier or excipient. Such carriers can be used to formulate tablets, powders, pills, troches, capsules, liquids, gels, syrups, elixirs, syrups, and suspensions for oral ingestion by a subject.

Pharmaceutical preparations for oral use can be obtained as follows: mixing one or more solid excipients with one or more compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries (if desired), to obtain tablets or dragee cores. The core may be provided with a suitable coating. For this purpose, concentrated sugar solutions may be used, which may contain excipients such as gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings, for example, to identify or characterize different combinations of active compound doses.

Pharmaceutical products that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. In some embodiments, the capsule comprises a hard gelatin capsule containing one or more of a drug, bovine and vegetable gelatin. Gelatin may be subjected to an alkaline treatment. Push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers may be added. All formulations for oral administration are provided in dosages suitable for such administration.

For buccal or sublingual administration, the composition may be a tablet, lozenge or gel.

Parenteral injections may be formulated for bolus injection or continuous infusion. The pharmaceutical compositions may be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds may be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. The suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, 0.9% saline or 5% aqueous dextrose, before use.

The active compounds can be administered topically, and can be formulated in a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, sticks, balms, creams, and ointments. Such pharmaceutical compositions may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

Formulations suitable for transdermal administration of the active compounds may employ transdermal delivery devices and transdermal delivery patches, and may be lipophilic emulsions or buffered aqueous solutions dissolved and/or dispersed in polymers or adhesives. Such patches may be constructed for continuous, pulsed or on-demand delivery of pharmaceutical compounds. Transdermal delivery may be accomplished by iontophoretic patches. In addition, transdermal patches can provide controlled delivery. The rate of absorption can be slowed by the use of a rate controlling membrane or by entrapping the compound within a polymer matrix or gel. Instead, absorption enhancers may be used to increase absorption. The absorption enhancer or carrier may include an absorbable pharmaceutically acceptable solvent to aid in penetration through the skin. For example, the transdermal device may be in the form of a bandage comprising a backing member, a reservoir containing the compound and a carrier, a rate controlling barrier for delivering the compound to the skin of the subject at a controlled and predetermined rate over an extended period of time, and an adhesive to secure the device to the skin or eye.

For administration by inhalation, the active compounds may be in the form of aerosols, mists or powders. The pharmaceutical compositions are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may also be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, gel suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, and synthetic polymers such as polyvinylpyrrolidone and PEG. In compositions in the form of suppositories, mixtures of low melting waxes such as fatty acid glycerides or cocoa butter may be used.

In practicing the treatment or methods of use provided herein, a therapeutically effective amount of a compound described herein is administered in the form of a pharmaceutical composition to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal, such as a human. The therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. These compounds may be used alone or as components of a mixture in combination with one or more therapeutic agents.

Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The formulation may be modified according to the chosen route of administration. Pharmaceutical compositions containing the compounds described herein may be prepared, for example, by mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing processes.

The pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier, diluent or excipient and a compound described herein in free base or pharmaceutically acceptable salt form. The methods and pharmaceutical compositions described herein include the use of crystalline forms (also referred to as polymorphs) and active metabolites of these compounds having the same type of activity.

Methods for preparing compositions comprising compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form solid, semi-solid, or liquid compositions. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include, for example, solutions in which the compounds are dissolved, emulsions comprising the compounds, or solutions containing liposomes, micelles, or nanoparticles comprising the compounds disclosed herein. Semi-solid compositions include, for example, gels, suspensions, and creams. The composition may be a liquid solution or suspension, a solid form suitable for dissolution or suspension in a liquid prior to use, or as an emulsion. These compositions may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and other pharmaceutically acceptable additives.

Non-limiting examples of dosage forms suitable for use herein include feeds, foods, pellets, lozenges, liquids, elixirs, aerosols, inhalants, sprays, powders, tablets, pills, capsules, gels, gelcaps (geltab), nanosuspensions, nanoparticles, microgels, suppository lozenges, aqueous or oily suspensions, ointments, patches, lotions, dentifrices, emulsions, creams, drops, dispersible powders or granules, emulsions in hard or soft gel capsules, syrups, botanicals (phytoceuticals), nutraceuticals, and any combination thereof.

Non-limiting examples of pharmaceutically acceptable excipients suitable for use herein include granulating agents, binders, lubricants, disintegrating agents, sweetening agents, glidants, anti-sticking agents, antistatic agents, surfactants, antioxidants, gums, coating agents, coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents, antimicrobial agents, plant cellulose materials and spheronizing agents and any combination thereof.

The compositions described herein may be, for example, in immediate release form or in controlled release formulation. Immediate release formulations may be formulated to allow the compound to act rapidly. Non-limiting examples of immediate release formulations include readily soluble formulations. Controlled release formulations may be pharmaceutical formulations that have been adjusted such that the drug release rate and drug release profile may match physiological and temporal therapeutic requirements, or that have been formulated to achieve release of the drug at a programmed rate. Non-limiting examples of controlled release formulations include particles, delayed release particles, hydrogels (e.g., of synthetic or natural origin), other gelling agents (e.g., gel-forming dietary fibers), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed therein), particles within a matrix, polymeric mixtures, and particulate agglomerates.

The disclosed compositions may optionally comprise from about 0.001% to about 0.005% weight/volume of a pharmaceutically acceptable preservative. One non-limiting example of a suitable preservative is benzyl alcohol.

In some embodiments, the controlled release formulation is a delayed release form. The delayed release form may be formulated to delay the action of the compound for an extended period of time. The delayed release form may be formulated to delay the release of the effective dose or doses of the compound, for example for about 4 hours, about 8 hours, about 12 hours, about 16 hours, or about 24 hours.

The controlled release formulation may be in a sustained release form. Sustained release forms may be formulated to maintain the effect of, for example, the compound over an extended period of time. Sustained release forms can be formulated to provide an effective dose of any of the compounds described herein (e.g., to provide a physiologically effective blood distribution) within about 4 hours, about 8 hours, about 12 hours, about 16 hours, or about 24 hours.

Non-limiting examples of pharmaceutically acceptable excipients can be found, for example, in Remington: the Science and practice of Pharmacy, 19 th edition (Easton, Pa.: Mack Publishing Company, 1995); hoover, John e., Remington's Pharmaceutical Sciences, Mack Publishing co, Easton, Pennsylvania 1975; liberman, h.a. and Lachman, l., eds., Pharmaceutical DosageForms, Marcel Decker, New York, n.y., 1980; and Pharmaceutical document Forms and drug delivery Systems, 7 th edition (Lippincott Williams & Wilkins1999), each of which is incorporated by reference in its entirety.

The disclosed methods comprise administering a decitabine derivative dinucleotide, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier. The carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

The compounds of formula (1) herein or pharmaceutically acceptable salts thereof may be conveniently formulated into pharmaceutical compositions consisting of one or more pharmaceutically acceptable carriers. See, e.g., Remington's Pharmaceutical Sciences, latest edition, e.w. martin Mack pub.co., Easton, PA, which discloses typical carriers and conventional methods for preparing Pharmaceutical compositions that can be used with formulations for preparing the compounds described herein, and are incorporated herein by reference. Such medicaments may be standard carriers for administration of the compositions to humans and non-humans, including solutions, such as saline and buffered solutions at physiological pH. Other compositions may be administered according to standard procedures. For example, the pharmaceutical composition may further comprise one or more additional active ingredients, such as antimicrobial agents, anti-inflammatory agents, and anesthetics.

Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, saline, ringer's solution, and dextrose solution. The pH of the solution may be from about 5 to about 8, and may be from about 7 to about 7.5. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing a compound of formula (1) or a pharmaceutically acceptable salt thereof, in the form of shaped articles, e.g., films, liposomes, microparticles or microcapsules.

The disclosed methods involve administering a compound of formula (1) or a pharmaceutically acceptable salt thereof as part of a pharmaceutical composition. In various embodiments, the compositions described herein may include liquids comprising the active agent in the form of a solution, a suspension, or both. The liquid composition may comprise a gel. In one embodiment, the liquid composition is aqueous. Alternatively, the composition may take the form of an ointment. In another embodiment, the composition is an in situ gellable aqueous composition. In some embodiments, the composition is an in situ gellable aqueous solution.

In addition to the compounds disclosed herein, the pharmaceutical formulations may contain additional carriers, as well as thickening agents, diluents, buffers, preservatives, and surfactants. The pharmaceutical formulation may also contain one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents and anesthetics.

The excipient may simply and directly function as an inert filler, or the excipient used herein may be part of a pH stabilizing system or coating to ensure that the ingredients are safely delivered to the stomach.

The compound of formula (1) or a pharmaceutically acceptable salt thereof may also be present in a liquid, emulsion or suspension for delivery of the active therapeutic agent in the form of an aerosol to a body cavity, such as the nasal, laryngeal or bronchial passages. The ratio of the compound of formula (1) or a pharmaceutically acceptable salt thereof to the other compounding agents in these preparations may vary depending on the requirements of the dosage form.

Depending on the intended mode of administration, the pharmaceutical compositions administered as part of the disclosed methods may be in the form of solid, semi-solid, or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, and the like, e.g., unit dosage forms suitable for single administration of precise dosages. As described above, the composition may contain an effective amount of the compound of formula (1) or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier, and may further comprise other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and the like.

For solid compositions, non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate.

Pharmaceutically acceptable salts

In each of the foregoing aspects and embodiments described herein, the compounds of formula (1) may be used in salt or non-salt form.

Pharmaceutically acceptable salts include, for example, acid addition salts and base addition salts. The acid added to the compound to form an acid addition salt may be an organic acid or an inorganic acid. The base added to the compound to form a base addition salt may be an organic base or an inorganic base. In some embodiments, the pharmaceutically acceptable salt is a metal salt. In some embodiments, the pharmaceutically acceptable salt is an ammonium salt.

Acid addition salts can be produced by adding an acid to a compound described herein. In some embodiments, the acid is an organic acid. In some embodiments, the acid is an inorganic acid. Non-limiting examples of suitable acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, phosphoric acid, nicotinic acid, isonicotinic acid, lactic acid, salicylic acid, 4-aminosalicylic acid, tartaric acid, ascorbic acid, gentisic acid, gluconic acid, glucuronic acid, saccharic acid (saccaric acid), formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, citric acid, oxalic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, glycolic acid, malic acid, cinnamic acid, mandelic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, pamoic acid, phenylacetic acid, N-cyclohexylsulfamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1, 2-disulfonic acid, 4-methylbenzenesulfonic acid, benzoic acid, naphthalene-2-sulfonic acid, naphthalene-1, 5-disulfonic acid, 2-phosphoglycerate, 3-phosphoglycerate, glucose-6-phosphate and amino acids.

Non-limiting examples of suitable acid addition salts include hydrochloride, hydrobromide, hydroiodide, nitrate, nitrite, sulfate, sulfite, phosphate, hydrogen phosphate, dihydrogen phosphate, carbonate, hydrogen carbonate, nicotinate, isonicotinate, lactate, salicylate, 4-aminosalicylate, tartrate, ascorbate, gentisate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, pantothenate, acetate, propionate, butyrate, fumarate, succinate, citrate, oxalate, maleate, hydroxymaleate, methylmaleate, glycolate, malate, cinnamate, mandelate, 2-phenoxybenzoate, 2-acetoxybenzoate, pamoate, phenylacetate, xylonate, n-cyclohexylsulfamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, ethane-1, 2-disulfonate, 4-methylbenzenesulfonate, naphthalene-2-sulfonate, naphthalene-1, 5-disulfonate, 2-phosphoglycerate, 3-phosphoglycerate, glucose-6-phosphate and amino acid salt.

The metal salt may be produced by adding an inorganic base to the compounds described herein. The inorganic base consists of a metal cation paired with a basic counterion such as hydroxide, carbonate, bicarbonate or phosphate. The metal may be an alkali metal, an alkaline earth metal, a transition metal or a main group metal. Non-limiting examples of suitable metals include lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, and zinc.

Non-limiting examples of suitable metal salts include lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium and zinc salts.

Ammonium salts can be produced by adding ammonia or an organic amine to the compounds described herein. Non-limiting examples of suitable organic amines include triethylamine, diisopropylamine, ethanolamine, diethanolamine, triethanolamine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrazole (pyrazole), prazole (pipyrazole), imidazole, pyrazine, pipyrazine, ethylenediamine, N' -dibenzylethylenediamine, procaine, chloroprocaine, choline, dicyclohexylamine, and N-methylglucamine.

Non-limiting examples of suitable ammonium salts include triethylamine, diisopropylamine, ethanolamine, diethanolamine, triethanolamine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrazole, prazole, imidazole, pyrazine, pipyrazine, ethylenediamine, N' -dibenzylethylenediamine, procaine, chloroprocaine, choline, dicyclohexylamine, and N-methylglucamine.

One specific example of the salt of the compound of formula (1) is a sodium salt.

Therapeutic uses

The lyophilized pharmaceutical compositions according to the present disclosure can be used to treat a number of diseases susceptible to treatment with decitabine, including those described herein.

Thus, in other aspects, the present disclosure provides: (i) a lyophilized pharmaceutical composition as described herein for use in medicine; (ii) a lyophilized pharmaceutical composition as described herein for use in the treatment of a disease as described herein; (iii) a method of treating a disease described herein, the method comprising mixing a lyophilized pharmaceutical composition described herein with a pharmaceutically acceptable solvent and administering an effective amount of the mixture to a subject in need thereof; (iv) use of a lyophilized pharmaceutical composition described herein for the manufacture of a medicament for the treatment of a disease described herein; (v) a method of treating cancer in a patient in need thereof, the method comprising reconstituting a lyophilized pharmaceutical composition described herein in a pharmaceutically acceptable solvent to obtain a liquid formulation comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof, and administering a therapeutically effective amount of the liquid formulation to the patient.

Examples of diseases that can be treated using the lyophilized pharmaceutical compositions of the present disclosure include those involving unwanted or uncontrolled cellular proliferation. Such indications include benign tumors, various types of cancer such as primary tumors and tumor metastases, restenosis (e.g., coronary, carotid, and brain lesions), hematologic disorders, abnormal stimulation of endothelial cells (atherosclerosis), injury to body tissue resulting from surgery, abnormal wound healing, abnormal angiogenesis, diseases that produce tissue fibrosis, repetitive movement disorders, disorders of non-highly vascularized tissue, and proliferative responses associated with organ transplantation.

Generally, cells in benign tumors retain their differentiated characteristics and do not divide in a completely uncontrolled manner. Benign tumors are usually localized and non-metastatic. Specific types of benign tumors that can be treated with the present disclosure include hemangioma, hepatocellular adenoma, cavernous hemangioma, focal nodular hyperplasia, acoustic neuroma, fibroma, cholangioadenoma, cholangiocystadenoma (cystanoma), fibroma, lipoma, leiomyoma, mesothelioma, teratoma, myxoma, nodular recurrent hyperplasia, trachoma, and pyogenic granuloma.

In malignant tumors, cells become undifferentiated, do not respond to the body's growth control signals, and proliferate in an uncontrolled manner. Malignant tumors are invasive and can spread to distant sites (metastases). Malignant tumors are generally divided into two categories: primary and secondary. Primary tumors arise directly from the tissues in which they are found. Secondary or metastatic tumors are tumors that originate elsewhere in the body but have now spread to distant organs. Common routes of metastasis are direct growth into adjacent structures, diffusion through the blood vessels or lymphatic system, and travel along tissue planes and body spaces (ascites, cerebrospinal fluid, etc.).

Examples of cancer are cancer, for example, bladder cancer, breast cancer, colon cancer, kidney cancer, epidermal cancer, liver cancer, lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, cancer of the gastrointestinal system or skin cancer, hematopoietic system tumors, such as leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, hairy cell lymphoma or burkitt's lymphoma; hematopoietic tumors of myeloid lineage, such as acute and chronic myelogenous leukemia, myelodysplastic syndrome, or promyelocytic leukemia; thyroid follicular cancer; tumors of mesenchymal origin, such as fibrosarcoma or rhabdomyosarcoma; tumors of the central or peripheral nervous system, such as astrocytomas, neuroblastoma, glioma or schwannoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum; keratoacanthoma (keratocotanthhoma); thyroid follicular cancer; or kaposi sarcoma.

Specific types of cancers or malignancies (primary or secondary) that can be treated using the compositions described herein include, for example, bladder cancer, breast cancer, ovarian cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, larynx cancer, gallbladder cancer, pancreatic cancer, rectal cancer, parathyroid cancer, thyroid cancer, adrenal cancer, neural tissue cancer, head and neck cancer, colon cancer, stomach cancer, bronchial cancer, kidney cancer, basal cell carcinoma, squamous cell carcinoma of the ulcerated and papillary types, metastatic skin cancer, osteosarcoma, ewing's sarcoma, reticulocytoma, myeloma, giant cell tumor, small cell lung tumor, gallstone, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuroma, enteric ganglioneuroma, cell tumor, Proliferative corneal neuromas, marfan's syndrome-like habit tumors, wilms ' tumors, seminoma, ovarian tumors, smooth muscle tumors, cervical dysplasia and carcinoma in situ, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid tumors, localized skin lesions, mycosis fungoides, rhabdomyosarcoma, kaposi's sarcoma, osteogenic and other sarcomas, malignant hypercalcemia, renal cell tumors, polycythemia vera, adenocarcinoma, glioblastoma multiforme, leukemia, lymphoma, malignant melanoma, epidermoid carcinoma, and other carcinomas and sarcomas.

In one embodiment, the cancer is selected from myelodysplastic syndrome, acute myelogenous leukemia, ovarian cancer, liver cancer, and colorectal cancer.

Hematologic disorders include abnormal growth of blood cells, which can lead to dysplastic changes in blood cells and hematologic malignancies, such as various leukemias. Examples of hematological disorders include, but are not limited to, acute myeloid leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, and sickle cell anemia.

The treatment of abnormal cell proliferation during surgery due to damage to body tissue may be used in a variety of surgical procedures, including joint surgery, bowel surgery, and keloid scarring. Diseases that produce fibrotic tissue include emphysema.

Repetitive motion disorders that can be treated with the present disclosure include carpal tunnel syndrome. One example of a cell proliferative disorder that can be treated with the present disclosure is bone tumors.

Proliferative responses associated with organ transplantation that may be treated with the present disclosure include those that promote potential organ rejection or related complications. In particular, these proliferative responses may occur during transplantation of the heart, lungs, liver, kidneys and other body organs or organ systems.

Angiogenic abnormalities that can be treated with the present disclosure include those associated with rheumatoid arthritis, ischemia reperfusion-associated cerebral edema and injury, cortical ischemia, ovarian hyperplasia and vascular hyperproliferation (polycystic ovary syndrome), endometriosis, psoriasis, diabetic retinopathy, and other ocular angiogenic diseases, such as retinopathy of prematurity (retrolental fibroplasia), muscle degeneration, corneal graft rejection, neovascular glaucoma (neovascular glaucoma), and ostwell syndrome (Oster Webber syndrome).

Diseases associated with abnormal angiogenesis require or induce blood vessel growth. For example, corneal angiogenesis includes three stages: pre-vascular latency, active neoangiogenesis, and vascular maturation and regression. The identity and mechanism of various angiogenic factors, including elements of the inflammatory response such as leukocytes, platelets, cytokines and eicosanoids or unidentified plasma components, remains to be revealed.

In some embodiments, the lyophilized pharmaceutical compositions of the present disclosure can be used to treat diseases associated with undesired or abnormal angiogenesis. The treatment methods can include administering to a patient suffering from undesired or abnormal angiogenesis a pharmaceutical formulation of the present disclosure, alone or in combination with an antineoplastic agent whose activity in vivo as an antineoplastic agent is adversely affected by high levels of DNA methylation. The specific dosage of these agents required to inhibit angiogenesis and/or angiogenic diseases may depend on the severity of the condition, the route of administration and related factors, which may be determined by the attending physician. Generally, an acceptable effective daily dose is an amount sufficient to effectively inhibit angiogenesis and/or angiogenic diseases.

The lyophilized pharmaceutical compositions of the present disclosure can be used to treat a variety of diseases associated with undesirable angiogenesis such as retinal/choroidal neovascularization and corneal neovascularization. Examples of retinal/choroidal neovascularization include, but are not limited to, Bestyes, myopia, optic fossa, Stargarts disease, Paget's disease, vein occlusion, artery occlusion, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum carotid obstructive disease (pseudoxanthoma elasticum obstructive diseases), chronic uveitis/vitritis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eales disease, diabetic retinopathy, macular degeneration, Bechets disease, infection causing retinitis or choroiditis (chroiitis), presumed ocular histoplasmosis, pars planaritis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma and post-laser complications, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by fibrovascular or fibrous tissue abnormal proliferation, including all forms of proliferative vitreoretinopathy. Examples of corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin a deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, synergestic syndrome (sjogrens), rosacea, vesicular disease (phylectenulosis), diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, silkworm-erosive corneal ulcer, Terrien limbic degeneration, limbic keratolysis, polyarteritis, wegener's sarcoidosis, scleritis, pemphigoid (periphigoid) radial keratotomy, neovascular glaucoma and retrolental fibrosis, syphilis, mycobacterial infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infections, herpes zoster infections, protozoal infections, and kaposi's sarcoma.

In some embodiments, the lyophilized pharmaceutical compositions of the present disclosure can be used to treat chronic inflammatory diseases associated with abnormal angiogenesis. The method comprises administering to a patient suffering from a chronic inflammatory disease associated with abnormal angiogenesis a pharmaceutical formulation of the present disclosure, alone or in combination with an antineoplastic agent whose activity in vivo as an antineoplastic agent is adversely affected by high levels of DNA methylation. This chronic inflammation depends on the continuous formation of capillary sprouts to maintain an influx of inflammatory cells. The influx and presence of inflammatory cells produces granulomas and thus maintains a chronic inflammatory state. Inhibition of angiogenesis by the pharmaceutical formulation of the present disclosure may prevent the formation of granulomas, thereby alleviating the condition. Examples of chronic inflammatory diseases include, but are not limited to, inflammatory bowel diseases such as crohn's disease and ulcerative colitis, psoriasis, sarcoidosis (sarcoidois), and rheumatoid arthritis.

Inflammatory bowel diseases such as crohn's disease and ulcerative colitis are characterized by chronic inflammation and angiogenesis at different sites in the gastrointestinal tract. For example, crohn's disease occurs as a chronic transmural inflammatory disease, most commonly affecting the terminal ileum and colon, but may also occur in any part of the gastrointestinal tract from the mouth to the anus and perianal region. Patients with crohn's disease often have chronic diarrhea, fever, anorexia, weight loss, and abdominal swelling associated with abdominal pain. Ulcerative colitis is also a chronic, non-specific, inflammatory and ulcerative disease that occurs in the colonic mucosa and is characterized by the presence of hemorrhagic diarrhea. These inflammatory bowel diseases are usually caused by chronic granulomatous inflammation throughout the gastrointestinal tract, involving new capillary sprouts surrounded by inflammatory cell cartridges. Inhibition of angiogenesis with the pharmaceutical formulations of the present disclosure will inhibit the formation of capillary sprouts and prevent the formation of granulomas. Inflammatory bowel disease also exhibits additional intestinal manifestations (manifestations), such as skin lesions. Such lesions are characterized by inflammation and angiogenesis and may occur in many locations outside the gastrointestinal tract. Inhibition of angiogenesis with the lyophilized pharmaceutical composition of the present disclosure will reduce the influx of inflammatory cells and prevent the formation of lesions.

Sarcoidosis, another chronic inflammatory disease, is characterized by multisystem granulomatous disease. The granuloma of the disease can form anywhere in the body, and thus, its symptoms depend on the site of the granuloma and whether the disease is active. Granulomas are produced by capillary sprouts that provide a stable supply of angiogenic blood vessels for inflammatory cells. Such granuloma formation can be inhibited by inhibiting angiogenesis using the lyophilized pharmaceutical composition of the present invention. Psoriasis, yet another chronic recurrent inflammatory disease, is characterized by papules and plaques of different sizes. Treatment with the pharmaceutical formulations of the present disclosure may reduce the likelihood of neovascularization necessary to maintain the characteristic lesion and result in a reduction in the patient's symptoms.

Rheumatoid Arthritis (RA) is also a chronic inflammatory disease characterized by nonspecific inflammation of peripheral joints. Blood vessels in the synovial lining of joints can undergo angiogenesis. In addition to forming a new vascular network, endothelial cells release factors and reactive oxygen species, leading to pannus growth and cartilage destruction. Factors involved in angiogenesis may contribute positively to and help maintain the chronic inflammatory state of rheumatoid arthritis. Treatment with the pharmaceutical formulations of the present disclosure, alone or in combination with other anti-RA agents, can reduce the likelihood of neovascularization necessary to maintain the chronic inflammation and provide relief from symptoms in RA patients.

the present invention relates to pharmaceutical compositions comprising decitabine and a pharmaceutically acceptable carrier, and more particularly to pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable carrier, excipient, carrier, diluent, excipient, carrier, excipient, diluent, excipient, carrier, diluent, excipient, carrier, excipient, or any combination thereof.

In some embodiments, the lyophilized pharmaceutical compositions of the present disclosure can be used to control gene expression within a cell. The method of treatment may comprise administering a pharmaceutical formulation of the present disclosure to a patient suffering from a disease associated with abnormal gene expression levels. DNA methylation is associated with the control of gene expression. Specifically, methylation in or near the promoter inhibits transcription, while demethylation restores expression. Examples of possible applications of the mechanism include, but are not limited to, therapeutically regulated growth inhibition, induction of apoptosis, and cell differentiation.

In some embodiments, the lyophilized pharmaceutical compositions of the present disclosure can be used to treat patients having a gene mutation associated with tumor hypermethylation, such as patients having a tumor type comprising a Succinate Dehydrogenase (SDH) mutation or defect, including patients having a gastrointestinal stromal tumor (GIST) that is not a KIT mutation.

The gene activation promoted by the lyophilized pharmaceutical composition of the present disclosure can induce differentiation of cells for therapeutic purposes. Cell differentiation is induced by a hypomethylation mechanism. Examples of morphological and functional differentiation include, but are not limited to, differentiation towards the formation of muscle cells, myotubes, erythroid and lymphoid lineage cells.

Myelodysplastic syndrome (MDS) is a heterogeneous clonal hematopoietic stem cell disorder associated with the presence of dysplastic changes of one or more hematopoietic lineages, including dysplastic changes of the myeloid, erythroid and megakaryocytic lineages. These changes result in a decrease in blood cells of one or more of these three lineages. Subjects with MDS typically develop complications associated with anemia, neutropenia (infection), or thrombocytopenia (hemorrhage). Typically, about 10% to about 70% of MDS subjects develop acute leukemia. Representative myelodysplastic syndromes include acute myeloid leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia, and chronic myelogenous leukemia.

Acute Myeloid Leukemia (AML) is the most common type of acute leukemia in adults. Several genetically acquired genetic disorders and immunodeficiency states are associated with increased risk of AML. These include disorders with defective DNA stability leading to random chromosome breakage, such as Bloom syndrome, Fanconi's anaemia, Li-Fraumeni family, ataxia telangiectasia and X-linked agammaglobulinemia.

Acute promyelocytic leukemia (APML) represents a distinct subpopulation of AML. This subtype is characterized by containing 15; 17 chromosome translocating promyelocytic meiocytes (blasts). This translocation results in the production of a fusion transcript comprising the retinoic acid receptor sequence and promyelocytic leukemia sequence.

Acute Lymphoblastic Leukemia (ALL) is a heterogeneous disease with unique clinical features exhibited by individual subtypes. Recurrent cytogenetic abnormalities have been demonstrated in ALL. The most common associated cytogenetic abnormalities are 9, which lead to the development of the philadelphia chromosome; 22 translocation.

In some embodiments, the lyophilized pharmaceutical compositions of the present disclosure can be used to treat MDS, e.g., MDS selected from AML, APML, and ALL.

For each of the foregoing therapeutic uses, the lyophilized pharmaceutical compositions of the present disclosure can be reconstituted in an appropriate solvent as described herein prior to administration to a subject, e.g., a mammalian subject, such as a human patient.

Administration and administration

A dose of a lyophilized pharmaceutical composition of the present disclosure (reconstituted or mixed with a pharmaceutically acceptable solvent or solvent mixture as described herein, as necessary) can be administered to a subject. Non-limiting examples of methods of administration include subcutaneous injection, intravenous injection, and infusion.

One dose of the formulation contains a therapeutically effective amount for treating the disease. A therapeutically effective amount of a compound of the present disclosure may be expressed as mg of the compound per kilogram of the subject's body weight. In some embodiments, the therapeutically effective amount is 1-1,000mg/kg, 1-500mg/kg, 1-250mg/kg, 1-100mg/kg, 1-50mg/kg, 1-25mg/kg, or 1-10 mg/kg. In some embodiments, the therapeutically effective amount is 5mg/kg, 10mg/kg, 25mg/kg, 50mg/kg, 75mg/kg, 100mg/kg, 150mg/kg, 200mg/kg, 250mg/kg, 300mg/kg, 400mg/kg, 500mg/kg, 600mg/kg, 700mg/kg, 800mg/kg, 900mg/kg, 1,000mg/kg, about 5mg/kg, about 10mg/kg, about 25mg/kg, about 50mg/kg, about 75mg/kg, about 100mg/kg, about 150mg/kg, about 200mg/kg, about 250mg/kg, about 300mg/kg, about 400mg/kg, about 500mg/kg, about 600mg/kg, about 700mg/kg, about 800mg/kg, about 900mg/kg or about 1,000 mg/kg.

The compounds described herein may be present in the composition as about 1mg to about 5mg, about 5mg to about 10mg, about 10mg to about 15mg, about 15mg to about 20mg, about 20mg to about 25mg, about 25mg to about 30mg, about 30mg to about 35mg, about 35mg to about 40mg, about 40mg to about 45mg, about 45mg to about 50mg, about 50mg to about 55mg, about 55mg to about 60mg, about 60mg to about 65mg, about 65mg to about 70mg, about 70mg to about 75mg, about 75mg to about 80mg, about 80mg to about 85mg, about 85mg to about 90mg, about 90mg to about 95mg, about 95mg to about 100mg, about 100mg to about 125mg, about 125mg to about 150mg, about 150mg to about 175mg, about 175mg to about 200mg, about 200mg to about 225mg, about 225mg to about 250mg, or about 250 mg.

The compound described herein may be present in the composition in an amount of about 1mg, about 5mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg, about 100mg, about 125mg, about 150mg, about 175mg, about 200mg, about 225mg, about 250mg, or about 300 mg.

In some embodiments, a therapeutically effective amount may be administered 1-35 times per week, 1-14 times per week, or 1-7 times per week. In some embodiments, a therapeutically effective amount may be administered 1-10 times per day, 1-5 times per day, 1,2, or 3 times per day.

The lyophilized pharmaceutical compositions described herein can be used alone or in combination with other chemotherapeutic agents or radiation therapy for the treatment of preventing or treating a range of proliferative disease states or conditions. Examples of such disease states and conditions are listed above.

The lyophilized pharmaceutical compositions of the present disclosure, administered alone or in combination with an anti-cancer agent and a therapy such as radiation therapy, can be administered to a subject, e.g., a human or animal patient, preferably a human, in need of such administration.

Examples of chemotherapeutic agents that may be co-administered with the lyophilized pharmaceutical compositions described herein include, but are not limited to, topoisomerase I inhibitors; other antimetabolites; a tubulin targeting agent; DNA binding agents and topoisomerase II inhibitors; an alkylating agent; a monoclonal antibody; an anti-hormone; a signal transduction inhibitor; a proteasome inhibitor; DNA methyltransferase inhibitors; a cytokine; an interferon; an interleukin; a retinoid; chromatin targeting therapies, such as HDAC or HAT modulators; t cell activators including immunomodulatory antibodies; a cancer vaccine; a hormonal drug; a plant-derived agent; a biological agent; an immunomodulator; radiotherapy; and other therapeutic or prophylactic agents; for example, agents that reduce or alleviate some of the side effects associated with chemotherapy; such as antiemetics and agents that prevent or reduce the duration of chemotherapy-associated neutropenia and prevent complications arising from reduced red blood cell or white blood cell levels, such as Erythropoietin (EPO), granulocyte macrophage colony stimulating factor (GM-CSF), and granulocyte colony stimulating factor (G-CSF).

In one embodiment, the lyophilized pharmaceutical compositions described herein are used in combination with an inhibitor of Histone Deacetylase (HDAC) to further modulate the transcription of genes in a synergistic manner, for example, to reconstitute the transcription of genes silenced by histone hypermethylation and acetylation.

Inhibitors of HDACs include, but are not limited to, the following structural classes: 1) hydroximic acid, 2) cyclic peptide, 3) benzamide, and 4) short chain fatty acid. Examples of hydroximic acids and hydroximic acid derivatives include trichostatin A (TSA), suberoylanilide hydroximic acid (SAHA), oxamflatin, suberic acid glyoxylic acid (SBHA), m-carboxy cinnamic acid glyoxylic acid (CBHA) and pyridoxamide (pyroxamide). TSA was isolated as an antifungal antibiotic and found to be a potent inhibitor of mammalian HDAC. The finding that TSA resistant cell lines have altered HDACs demonstrates that this enzyme is an important target for TSA. Other hydroxamic acid based HDAC inhibitors, SAHA, SBHA and CBHA, are synthetic compounds that are capable of inhibiting HDACs at micromolar concentrations or lower in vitro or in vivo. These hydroxamic acid based HDAC inhibitors all have basic structural features: a polar hydroxamic acid terminus linked through a hydrophobic methylene spacer (e.g., 6 carbons in length) to another polar site attached to a terminal hydrophobic moiety (e.g., a benzene ring).

The cyclic peptide used as HDAC inhibitor may be a cyclic tetrapeptide. Examples of cyclic peptides include, but are not limited to, trapoxin a, apicidin, and FR 901228. Trapoxin A is a cyclic tetrapeptide containing a 2-amino-8-oxo-9, 10-epoxydecanoyl (AOE) moiety. Apicidin is a fungal metabolite that exhibits potent broad-spectrum antiprotozoal activity and inhibits HDAC activity at nanomolar concentrations. FR901228 is a depsipeptide isolated from rhodobacter cyaneus (Chromobacterium violacea) and has been shown to inhibit HDAC activity at micromolar concentrations.

Examples of benzamides include, but are not limited to, MS-27-275. Examples of short chain fatty acids include, but are not limited to, butanoates (e.g., butyric acid, arginine butyrate, and Phenyl Butyrate (PB)). In addition, depudecin, which has been shown to inhibit HDAC at micromolar concentrations, can also be used in combination with the compositions disclosed herein.

In one embodiment, an alkylating agent is used in combination with the lyophilized pharmaceutical composition of the present invention. Examples of alkylating agents include the bis-chloroethylamines (nitrogen mustards, such as chlorambucil, cyclophosphamide, ifosfamide, nitrogen mustards, melphalan, uracil mustard), aziridines (such as thiotepa), alkyl ketosulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, streptozotocin), non-classical alkylating agents (hexamethylmelamine, dacarbazine and procarbazine) and platinum compounds (carboplatin and cisplatin).

In some embodiments, the lyophilized pharmaceutical compositions described herein can be used in combination with a platinum compound, such as cisplatin or carboplatin.

In some embodiments, the lyophilized pharmaceutical compositions described herein may be used in combination with a member of the retinoid superfamily, such as all-trans retinol, all-trans retinoic acid (tretinoin), 13-cis retinoic acid (isotretinoin), and 9-cis-retinoic acid.

In some embodiments, the lyophilized pharmaceutical compositions described herein may be used in combination with hormonal drugs such as synthetic estrogens (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen, toremifene, fluoxymesterone (fluoxymesterol), and raloxifene), antiandrogens (bicalutamide, nilutamide, flutamide), aromatase inhibitors (e.g., aminoglutethimide, anastrozole, and tetrazole), ketoconazole, goserelin acetate, leuprorelin, megestrol acetate, and mifepristone.

In some embodiments, the lyophilized pharmaceutical compositions described herein can be used in combination with a plant-derived agent such as vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinzolidine, and vinorelbine), camptothecins (20(S) -camptothecin, 9-nitro-20 (S) -camptothecin, and 9-amino-20 (S) -camptothecin), podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)), and taxanes (e.g., paclitaxel and docetaxel).

In some embodiments, the lyophilized pharmaceutical compositions described herein may be used in combination with a taxane such as paclitaxel and docetaxel.

In some embodiments, the lyophilized pharmaceutical compositions described herein may be used in combination with an anthracycline, such as daunorubicin or idarubicin.

In some embodiments, the lyophilized pharmaceutical compositions described herein can be used in combination with a biological agent such as an immunomodulatory protein (e.g., a cytokine), a monoclonal antibody against a tumor antigen, a tumor suppressor gene, or a cancer vaccine.

examples of interleukins that can be used in combination with the lyophilized pharmaceutical compositions disclosed herein include, but are not limited to, interleukin 2(IL-2) and interleukin 4(IL-4), interleukin 12(IL-12) examples of interferons that can be used with the lyophilized pharmaceutical compositions described herein include, but are not limited to, interferon [ β ], interferon [ beta ] (fibroblast interferon), and interferon [ gamma ] (fibroblast interferon).

Examples of monoclonal antibodies to tumor antigens that may be used with the lyophilized pharmaceutical compositions described herein include, but are not limited to, HERCEPTIN (R) (trastuzumab), RITUXAN (R) (Rituximab), MYLOTARG (R) (anti-CD 33), and CAMPATH (R) (anti-CD 52).

In some embodiments, the lyophilized pharmaceutical compositions described herein may be used in combination with a cancer vaccine, e.g., selected from a CTA cancer vaccine, such as a vaccine based on a CTA antigen selected from: NY-ESO-1, LAGE-1, MAGE-A1, -A2, -A3, -A4, -A6, -A10, -A12, CT7, CT10, GAGE1-6, GAGE 1-2, BAGE, SSX1-5, SSX2, HAGE, PRAME, RAGE-1, XAGE-1, MUC2, MUC5B, and HMW-MAA. Non-limiting examples of CTA vaccines include those based on MAGE-A3 (e.g., recMAGE-A3), NY-ESO-1 and PRAME.

In some embodiments, the lyophilized pharmaceutical compositions described herein may be used in combination with a T cell activator (e.g., a T cell activator that is an antibody (optionally a mAb)), for example selected from the group consisting of: (a) a CD137 agonist; (b) a CD40 agonist; (c) an OX40 agonist; (d) PD-1 mAb; (e) PD-L1 mAb; (f) CTLA-4 mAb; and (g) combinations of (a) - (f). In some embodiments, the adjunctive therapeutic component is tremelimumab or ipilimumab.

In some embodiments, the lyophilized pharmaceutical compositions described herein may be used in combination with carboplatin for the treatment of platinum-resistant recurrent ovarian cancer.

In some embodiments, the lyophilized pharmaceutical compositions described herein can be used to treat hepatocellular carcinoma (e.g., following failure of sorafenib treatment).

In some embodiments, the lyophilized pharmaceutical compositions described herein can be used in combination with irinotecan for the treatment of metastatic colon cancer.

In some embodiments, the lyophilized pharmaceutical compositions described herein can be used in combination with 5-fluorouracil (5-FU), leuocovorin, oxaliplatin for the treatment of metastatic colon cancer.

In some embodiments, the lyophilized pharmaceutical compositions described herein may be used in combination with cytarabine and fludarabine for the treatment of pediatric relapsed/refractory AML.

In some embodiments, the lyophilized pharmaceutical compositions described herein may be used in combination with a JAK2 inhibitor for the treatment of myeloproliferative neoplasms (myeloproliferative neoplasms).

The lyophilized pharmaceutical composition described herein and any other therapeutic agent may be provided separately or together in a pharmaceutical package, kit, or patient pack.

The lyophilized pharmaceutical compositions described herein, as well as combinations with other therapeutic agents or radiation therapies described above, can be administered over an extended period of time to maintain a beneficial therapeutic effect, or can be administered for only a short period of time. Alternatively, the compositions and combinations may be administered in a pulsed or continuous manner.

The lyophilized pharmaceutical compositions described herein can be administered in an effective amount, i.e., an amount effective to produce the desired therapeutic effect, either alone (monotherapy) or in combination with one or more chemotherapeutic agents or radiation therapy. For example, an effective amount can be an amount of a compound that, when administered to a subject having cancer, slows tumor growth, ameliorates disease symptoms, and/or prolongs lifespan.

The amount of the lyophilized pharmaceutical composition described herein administered to a subject can depend on the type and severity of the disease or condition as well as the characteristics of the subject, such as general health, age, sex, body weight, and tolerance to drugs. The skilled person will be able to determine the appropriate dosage in view of these and other factors.

Purity of the compounds disclosed herein

Any of the compounds herein can be purified. The compounds herein can be at least 1% pure, at least 2% pure, at least 3% pure, at least 4% pure, at least 5% pure, at least 6% pure, at least 7% pure, at least 8% pure, at least 9% pure, at least 10% pure, at least 11% pure, at least 12% pure, at least 13% pure, at least 14% pure, at least 15% pure, at least 16% pure, at least 17% pure, at least 18% pure, at least 19% pure, at least 20% pure, at least 21% pure, at least 22% pure, at least 23% pure, at least 24% pure, at least 25% pure, at least 26% pure, at least 27% pure, at least 28% pure, at least 29% pure, at least 30% pure, at least 31% pure, at least 32% pure, at least 33% pure, at least 34% pure, at least 35% pure, at least 36% pure, at least 37% pure, at least 38% pure, at least 39% pure, at least 40% pure, at least 41% pure, at least 42% pure, at least 11% pure, at least, At least 43% pure, at least 44% pure, at least 45% pure, at least 46% pure, at least 47% pure, at least 48% pure, at least 49% pure, at least 50% pure, at least 51% pure, at least 52% pure, at least 53% pure, at least 54% pure, at least 55% pure, at least 56% pure, at least 57% pure, at least 58% pure, at least 59% pure, at least 60% pure, at least 61% pure, at least 62% pure, at least 63% pure, at least 64% pure, at least 65% pure, at least 66% pure, at least 67% pure, at least 68% pure, at least 69% pure, at least 70% pure, at least 71% pure, at least 72% pure, at least 73% pure, at least 74% pure, at least 75% pure, at least 76% pure, at least 77% pure, at least 78% pure, at least 79% pure, at least 80% pure, at least 81% pure, at least 82% pure, at least 83% pure, at least 84% pure, at least 85% pure, at least, At least 86% pure, at least 87% pure, at least 88% pure, at least 89% pure, at least 90% pure, at least 91% pure, at least 92% pure, at least 93% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.1% pure, at least 99.2% pure, at least 99.3% pure, at least 99.4% pure, at least 99.5% pure, at least 99.6% pure, at least 99.7% pure, at least 99.8% pure, or at least 99.9% pure.

Impurities in the lyophilized pharmaceutical compositions described herein

Impurities may be formed, for example, by epimerization of the anomeric stereocenter in the decitabine fragment, synthetic by-products, degradation products, opening of the triazine ring with water, basic cleavage of the intermediate carboxamide after opening of the triazine ring with water, formation of a protected dimer and subsequent cleavage of the protecting group, or incomplete deprotection of the synthetic intermediate.

The lyophilized pharmaceutical compositions of the present disclosure may comprise an impurity, such as a nucleotide, a nucleoside, a compound comprising a ribose core, a compound comprising a deoxyribose core, a compound comprising a deoxyribonucleoside, or a compound comprising deoxyadenosine, wherein the impurity is not, for example, a compound of formula (1). In some embodiments, the lyophilized pharmaceutical compositions of the present disclosure comprise a compound comprising deoxyribose, a nitrogenous base (e.g., adenine), and a phosphate group, wherein the impurity is not, for example, a compound of formula (1).

Non-limiting examples of impurities in the lyophilized compositions of the present disclosure include compounds of formula (2):

or a pharmaceutically acceptable salt thereof, wherein the compound of formula (2) is not a compound of formula (1), wherein:

R¹is heteroaryl or urea, each of which is independently substituted or unsubstituted;

each R²And R³Independently substituted or unsubstituted alkyl; or hydrogen; and is

R⁴Is hydrogen or acyl, each of which is independently substituted or unsubstituted.

In some embodiments, R¹Is a substituted urea. In some embodiments, R¹Is urea substituted by methane diamine. In some embodiments, R¹Is urea substituted with N- (aminomethyl) formamide. In some embodiments, R¹Is heteroaryl. In some embodiments, R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

In some embodiments, each R is²And R³Independently hydrogen. In some embodiments, R²Is H and R³Is alkyl substituted by hydroxy. In some embodiments, R²Is H and R³Is an alkyl group substituted by an alkoxy group. In some embodiments, R²Is H and R³Is methyl substituted by methoxy. In some embodiments, R⁴Is hydrogen. In some embodiments, R⁴Is acyl, such as acetyl.

In some embodiments, the impurities in the lyophilized compositions of the present disclosure comprise formula (la)

(3) A compound:

or a pharmaceutically acceptable salt thereof, wherein R¹Is heteroaryl or urea, each of which is independently substituted or unsubstituted.

In some embodiments, R¹Is heteroaryl, e.g. 4-amino-2H-1 lambda ²3, 5-triazin-2-one. In some embodiments, R¹Are substituted ureas, for example ureas substituted with methane diamine.

In some embodiments, the impurities in the lyophilized compositions of the present disclosure comprise formula (la)

(4) A compound:

or a pharmaceutically acceptable salt thereof,

wherein:

R¹is a substituted or unsubstituted heteroaryl; and is

R⁵Is hydroxyl or nucleotide.

In some embodiments, R¹Is heteroaryl, e.g. 4-amino-2H-1 lambda ²3, 5-triazin-2-one or 2-amino-9 lambda²-purin-6 (1H) -one. In some embodiments, R⁵Is a hydroxyl group. In some embodiments, R⁵Is a nucleotide, for example of the formula:

in some embodiments, the impurity is a compound of the formula:

or a pharmaceutically acceptable salt thereof.

The impurities in the lyophilized composition may be present in an amount of up to about 0.01%, up to about 0.02%, up to about 0.03%, up to about 0.04%, up to about 0.05%, up to about 0.06%, up to about 0.07%, up to about 0.08%, up to about 0.09%, up to about 0.1%, up to about 0.12%, up to about 0.14%, up to about 0.16%, up to about 0.18%, up to about 0.2%, up to about 0.22%, up to about 0.24%, up to about 0.26%, up to about 0.28%, up to about 0.3%, up to about 0.32%, up to about 0.34%, up to about 0.36%, up to about 0.38%, up to about 0.4%, up to about 0.42%, up to about 0.44%, up to about 0.46%, up to about 0.48%, or up to about 0.5% of the lyophilized composition. In some embodiments, the impurities may be present in the lyophilized composition in an amount from about 0.05% to about 0.1%. In some embodiments, the impurities may be present in the lyophilized composition in an amount from about 0.05% to about 0.2%. In some embodiments, the impurities may be present in the lyophilized composition in an amount from about 0.05% to about 0.3%. In some embodiments, the impurities may be present in the lyophilized composition in an amount from about 0.05% to about 0.35%.

In some embodiments, the impurities may be present in the lyophilized composition in an amount of about 0.05%. In some embodiments, the impurities may be present in the lyophilized composition in an amount of about 0.1%. In some embodiments, the impurities may be present in the lyophilized composition in an amount of about 0.15%.

The lyophilized composition may comprise more than one impurity. For example, the lyophilized composition may comprise 1,2, 3,4, 5,6, 7,8, 9, or 10 impurities. In some embodiments, the lyophilized composition may comprise 3 impurities. In some embodiments, the lyophilized composition may comprise 4 impurities. In some embodiments, the lyophilized composition may comprise 5 impurities. In some embodiments, the lyophilized composition may comprise 6 impurities. In some embodiments, the lyophilized composition may comprise 7 impurities.

The ratio of compound of formula (1) to impurity in the pharmaceutical compositions of the present disclosure may be, for example, about 20,000: about 1, about 19,000: about 1, about 18,000: about 1, about 17,000: about 1, about 16,000: about 1, about 15,000: about 1, about 14,000: about 1, about 13,000: about 1, about 12,000: about 1, about 11,000: about 1, about 10,000: about 1, about 9,900: about 1, about 9,800: about 1, about 9,700: about 1, about 9,600: about 1, about 9,500: about 1, about 9,400: about 1, about 9,300: about 1, about 9,200: about 1, about 9,100: about 1, about 9,000: about 1, about 8,900: about 1, about 8,800: about 1, about 8,700: about 1, about 8,600: about 1, about 8,100: about 1, about 7: about 7,500: about 1, about 7,100: about 1, about 7: about 1, about 7,000: about 1, about 7,600: about 1, about 8,500: about 1, about 7,600: about 1, about 1,600: about 1, about 1, About 7,200: about 1, about 7,100: about 1, about 7,000: about 1, about 6,900: about 1, about 6,800: about 1, about 6,700: about 1, about 6,600: about 1, about 6,500: about 1, about 6,400: about 1, about 6,300: about 1, about 6,200: about 1, about 6,100: about 1, about 6,000: about 1, about 5,900: about 1, about 5,800: about 1, about 5,700: about 1, about 5,600: about 1, about 5,500: about 1, about 5,400: about 1, about 5,300: about 1, about 5,200: about 1, about 5,100: about 1, about 5,000: about 1, about 4,900: about 1, about 4,800: about 1, about 4,700: about 1, about 4,600: about 1, about 4,500: about 1, about 3: about 1, about 3,500: about 1, about 3,1, about 1, about 3: about 1,200: about 1, about 3,200: about 1, about 3,500: about 1, about 3,1, about 3,200: about 1, about 3,200: about 1, about 3,200: about 1, about 1, About 2,900: about 1, about 2,800: about 1, about 2,700: about 1, about 2,600: about 1, about 2,500: about 1, about 2,400: about 1, about 2,300: about 1, about 2,200: about 1, about 2,100: about 1, about 2,000: about 1, about 1,900: about 1, about 1,800: about 1, about 1,700: about 1, about 1,600: about 1, about 1,500: about 1, about 1,400: about 1, about 1,300: about 1, about 1,200: about 1, about 1,100: about 1, about 1,000: about 1, about 990: about 1, about 980: about 1, about 970: about 1, about 960: about 1, about 950: about 1, about 800: about 1, about 700: about 1, about 600: 1, about 500: about 1, about 300: about 1, about 1: about 1, about 1,80: about 1, about 1: about 80: about 1, about, About 45: about 1, about 40: about 1, about 35: about 1, about 30: about 1, about 25: about 1, about 20: about 1, about 19: about 1, about 18: about 1, about 17: about 1, about 16: about 1, about 15: about 1, about 14: about 1, about 13: about 1, about 12: about 1, about 11: about 1, or about 10: about 1.

The amount of an impurity in a composition of the present disclosure may be, for example, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3.3%, about 4.3%, about 4.4%, about 3.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.3%, about 3.1%, about 3.4%, about 4%, about 3.5%, about 4%, about 3.6%, about 4%, about 3.7%, about 4%, about 3.8%, about 4%, about 3%, about 4%, about 3.4%, about 4.9%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

The amount of impurity 1 in the compositions of the present disclosure may be, for example, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 4.3%, about 3.4%, about 3.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.3%, about 4%, about 3.4%, about 4%, about 3.5%, about 4%, about 3.6%, about 4%, about 3.7%, about 4%, about 3.4%, about 3%, about 4%, about 3%, about 4%, about 4.8%, about 4.9%, or about 5%. The amount of impurity 1 in the compositions of the present disclosure may range, for example, from about 0.01% to about 0.02%, 0.01% to about 0.03%, about 0.01% to about 0.04%, about 0.01% to about 0.05%, about 0.01% to about 0.08%, about 0.01% to about 0.1%, about 0.02% to about 0.03%, about 0.02% to about 0.04%, about 0.02% to about 0.05%, about 0.02% to about 0.08%, about 0.02% to about 0.1%, about 0.03% to about 0.04%, about 0.03% to about 0.05%, about 0.03% to about 0.06%, about 0.03% to about 0.1%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 1.5%, about 2% to about 5%, about 3.5% to about 4%, about 3.5%, about 3% to about 4.5%, about 3.5% to about 4%. In some embodiments, the amount of impurity 1 in the compositions disclosed herein is less than or equal to about 0.05%.

The amount of impurity 2 in the compositions of the present disclosure may be, for example, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.2%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 3.3%, about 2.3%, about 3.3%, about 2.3.3%, about 3.3%, about 3.5%, about 1.6%, about 1.7%, about 2.8%, about 2%, about 3.9%, about 2.3%, about 3%, about 3.3%, about 3%, about 3.5%, about 3%, about 3.2%, about 3%, about 3.8%, about 3.9%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, or about 5%. The amount of impurity 2 in the compositions of the present disclosure may range, for example, from about 0.05% to about 0.06%, from about 0.05% to about 0.07%, from about 0.05% to about 0.08%, from about 0.05% to about 0.09%, from about 0.05% to about 0.1%, from about 0.1% to about 0.2%, from about 0.1% to about 0.5%, from about 0.2% to about 0.3%, from about 0.5% to about 1%, from about 1% to about 1.5%, from about 1.5% to about 2%, from about 2% to about 2.5%, from about 2.5% to about 3%, from about 3% to about 3.5%, from about 3.5% to about 4%, from about 4% to about 4.5%, and from 4.5% to about 5%.

The amount of impurity 3 in the compositions of the present disclosure may be, for example, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3.3%, about 4.3%, about 3.4%, about 3.5%, about 3.6%, about 4.7%, about 3.8%, about 3%, about 4.9%, about 3%, about 3.4%, about 4%, about 3.5%, about 4%, about 3.6%, about 4%, about 3.6%, about 4%, about 3%, about 4%, about 3.7%, about 4%, about 3.9%, about 3%, about 4%, about 3, About 4.8%, about 4.9%, or about 5%. The amount of impurity 3 in the compositions of the present disclosure may range, for example, from about 0.01% to about 0.02%, 0.01% to about 0.03%, about 0.01% to about 0.04%, about 0.01% to about 0.05%, about 0.01% to about 0.08%, about 0.01% to about 0.1%, about 0.02% to about 0.03%, about 0.02% to about 0.04%, about 0.02% to about 0.05%, about 0.02% to about 0.08%, about 0.02% to about 0.1%, about 0.03% to about 0.04%, about 0.03% to about 0.05%, about 0.03% to about 0.06%, about 0.03% to about 0.1%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1.5% to about 1.5%, about 2% to about 5%, about 3.5% to about 4%, about 3.5% to about 4% to about 5%. In some embodiments, the amount of impurity 3 in the compositions disclosed herein is less than or equal to about 0.05%. In some embodiments, the amount of impurity 3 in the compositions disclosed herein is about 0.08%.

The amount of impurity 4 in the compositions of the present disclosure may be, for example, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about 4.3%, about 3.4%, about 3.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3.3%, about 4.3%, about 4%, about 3.3.3%, about 4%, about 3.5%, about 4%, about 3.6%, about 4%, about 3.7%, about 4%, about 3%, about 3.6%, about 4%, about 3%, about 4%, about 3., About 4.8%, about 4.9%, or about 5%. The amount of impurity 4 in the compositions of the present disclosure may range, for example, from about 0.01% to about 0.02%, 0.01% to about 0.03%, about 0.01% to about 0.04%, about 0.01% to about 0.05%, about 0.01% to about 0.08%, about 0.01% to about 0.1%, about 0.02% to about 0.03%, about 0.02% to about 0.04%, about 0.02% to about 0.05%, about 0.02% to about 0.08%, about 0.02% to about 0.1%, about 0.03% to about 0.04%, about 0.03% to about 0.05%, about 0.03% to about 0.06%, about 0.03% to about 0.1%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1.5% to about 1.5%, about 2% to about 5%, about 3.5% to about 3.5%, about 3.5% to about 4%, about 3.5%, about 3% to about 4% to about 5%. In some embodiments, the amount of impurity 4 in the compositions disclosed herein is less than or equal to about 0.05%. In some embodiments, the amount of impurity 4 in the compositions disclosed herein is about 0.06%.

The amount of impurity 5 in the compositions of the present disclosure may be, for example, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.2%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 3.3%, about 2.3%, about 3.3%, about 2.3.3%, about 3.3%, about 2%, about 3.3%, about 3%, about 2.3%, about 3%, about 3.5%, about 3%, about 3.2%, about 3%, about 3.2%, about 3%, about, About 3.8%, about 3.9%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, or about 5%. The amount of impurity 5 in the compositions of the present disclosure may range, for example, from about 0.05% to about 0.06%, from about 0.05% to about 0.07%, from about 0.05% to about 0.08%, from about 0.05% to about 0.09%, from about 0.05% to about 0.1%, from about 0.1% to about 0.2%, from about 0.1% to about 0.5%, from about 0.2% to about 0.3%, from about 0.5% to about 1%, from about 1% to about 1.5%, from about 1.5% to about 2%, from about 2% to about 2.5%, from about 2.5% to about 3%, from about 3% to about 3.5%, from about 3.5% to about 4%, from about 4% to about 4.5%, and from 4.5% to about 5%.

The amount of impurity 6 in the compositions of the present disclosure may be, for example, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.2%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 3.3%, about 2.3%, about 3.3%, about 2.3.3%, about 3.3%, about 2%, about 3.3%, about 3.5%, about 3.7%, about 2%, about 3%, about 3.3%, about 3%, about 3.5%, about 2.2%, about 3%, about 3.2%, about 3, About 3.8%, about 3.9%, about 4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, or about 5%. The amount of impurity 6 in the compositions of the present disclosure may range, for example, from about 0.05% to about 0.06%, from about 0.05% to about 0.07%, from about 0.05% to about 0.08%, from about 0.05% to about 0.09%, from about 0.05% to about 0.1%, from about 0.1% to about 0.2%, from about 0.1% to about 0.5%, from about 0.2% to about 0.3%, from about 0.5% to about 1%, from about 1% to about 1.5%, from about 1.5% to about 2%, from about 2% to about 2.5%, from about 2.5% to about 3%, from about 3% to about 3.5%, from about 3.5% to about 4%, from about 4% to about 4.5%, and from 4.5% to about 5%.

Non-limiting examples of methods that can be used to identify impurities of the present disclosure include High Performance Liquid Chromatography (HPLC), Mass Spectrometry (MS), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), electrospray ionization time-of-flight (ESI-TOF), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and two-dimensional gel electrophoresis.

HPLC can use high pressure to separate components of a mixture through a packed column of solid adsorbent material (denoted as the stationary phase) to identify impurities. Sample components may interact differently with the column depending on the pressure applied to the column, the material used in the stationary phase, the size of the particles used in the stationary phase, the composition of the solvent used in the column, and the temperature of the column. The interaction between the sample component and the stationary phase can affect the time required for the sample component to travel through the column. The time required for the components to travel from the injection point to the elution through the column is called the retention time.

After elution from the column, the eluted components may be detected using an ultraviolet detector attached to the column. The wavelength of light at which a component is detected, in combination with the retention time of the component, can be used to identify the component. In addition, the peaks displayed by the detector can be used to determine the amount of the component present in the initial sample. Wavelengths of light that can be used to detect components of the sample include, for example, about 200nM, about 225nM, about 250nM, about 275nM, about 300nM, about 325nM, about 350nM, about 375nM, and about 400 nM.

Mass Spectrometry (MS) can also be used to identify impurities of the compounds of the present disclosure. To prepare samples for MS analysis, a sample containing the protein of interest is digested into smaller peptides with proteolytic enzymes. The enzyme used for cleavage may be, for example, trypsin, chymotrypsin, glutamyl endopeptidase, Lys-C and pepsin. The sample may be injected into the mass spectrometer. After implantation, all or most of the impurities can be ionized and detected as spectrally-oriented ions, depending on the mass-to-charge ratio produced upon ionization. The mass to charge ratio can then be used to determine the impurities present in the sample.

The present disclosure provides several embodiments of pharmaceutical formulations that have advantages in terms of stability, administration, efficacy, and modulation of formulation viscosity. Any of the embodiments disclosed herein may be used together or separately. For example, any of the pharmaceutically acceptable excipients, methods, techniques, solvents, or compounds disclosed herein may be used with any other pharmaceutically acceptable excipient, method, technique, solvent, or compound disclosed herein to achieve any therapeutic result. The compounds, excipients, and other formulation components may be present in any such formulation in any amount, proportion, or percentage disclosed herein, and any such combination may be used therapeutically for any purpose described herein and to provide any viscosity described herein.

Examples

Example 1 preparation of a lyophilized formulation of the sodium salt of the compound of formula (1).

The sodium salt of the compound of formula (1) was dissolved in DMSO at a defined concentration using an overhead mixer in an appropriately sized Stainless Steel (SS) vessel. After complete dissolution of the drug in DMSO, a sample of the bulk solution was assayed using UV or HPLC in-process methods to determine the amount of the sodium salt of the compound of formula 1 to be within 95-105% of the target concentration. The bulk solution was filtered through a series of two pre-sterilized 0.2 micron sterile filters compatible with DMSO and collected into 2L SS buffer containers. By visual monitoring of the amount available for filling the buffer containerThe filtration rate is adjusted. A1 gram aliquot of the filtered bulk solution was then filled into a 5cc depyrogenated clear glass vial. Each vial was automatically and partially stoppered on a filling line with a pre-sterilized fluoropolymer coated chlorobutyl rubber lyo stopper. The product vials were transferred to a lyophilizer under sterile transfer conditions to initiate the lyophilization cycle. The freeze-dryer used was a pilot-scale freeze-dryer Lyobeta 35, IMA-Telstar, with 1.02m²A cavity space of 35kg of ice capacity, a condenser capacity of 22kg/24 hr.

The general lyophilization cycle for the compound of formula (1) is:

1. the shelves were controlled at a target set point of 20 ℃ until product was loaded onto the shelves. The temperature was held for 1 hour to allow all product samples to equilibrate at the target temperature.

2. The shelves were cooled at an average controlled rate of 30 ℃/hour to a target shelf set point of-45 ℃. The target shelf set point was maintained for 1 hour to allow all product to equilibrate and fully cure at the target temperature.

3. The shelves were ramped up to a target shelf temperature set point of 0 ℃ at an average controlled rate of 30 ℃/hour. The target shelf temperature was maintained at the set point for 2 hours to allow all product samples to anneal at the target temperature.

4. The shelves were cooled at an average controlled rate of 30 ℃/hour to a target shelf set point of-45 ℃. The target shelf set point was maintained for 2 hours to allow all product samples to equilibrate and fully cure at the target temperature.

5. The condenser was cooled to below-40 ℃ and the chamber was evacuated to the target pressure. The target shelf set point was held for an additional 4 hours to allow any unfrozen DMSO to evaporate.

6. The chamber pressure was controlled at a target set point to sublime the DMSO.

7. The shelves were ramped up to a target shelf temperature set point of-6 ℃ at an average controlled rate of 30 ℃/hour and controlled at the target shelf set point for 80.5 hours until all DMSO had sublimated.

8. The shelves were ramped up at an average controlled rate to a target shelf temperature setpoint of 40 ℃ and held at the target shelf setpoint to reduce the residual DMSO level.

9. The shelf was cooled to a target set point of 20 ℃ for unloading. The chamber pressure was raised to 14.7 ± 0.7PSIA by charging the chamber with filtered nitrogen NF. The vial was stoppered and unloaded.

Specific lyophilization parameters for this study are provided in table 1 below:

the thermocouple and pressure results of the foregoing lyophilization cycle of table 1 are shown in fig. 2.

A summary of the product temperatures at equilibrium of the foregoing lyophilization cycle parameters is shown in table 2 below:

¹the product temperature indicates the temperature at the end of the run.

²Indicating the temperature of the product immediately prior to evacuation.

A summary of the product off temperatures is shown in table 3 below.

After lyophilization, the product appeared as a dense white cake, as shown in fig. 3. The original filling height was 5-6mm and the product height was 4mm, with uniform shrinkage observed around the 1mm side. The top of the cake appeared matte with some areas glossy, while the sides and bottom appeared glossy. The top of the cake was concave and had streaks and cracks in the texture. Upon inversion, the cake remained intact and moved to the top of the vial. Upon shaking, the cake moved to the top of the vial and broke into pieces and powder. There was a minimal amount of residual material around the original fill level, as a viscous white film.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. Because of the long reconstitution time, the reconstitution time for this study is reported in minutes, as shown in table 4 below:

figure 4 provides an overlay of the TGA results of this lyophilization and study with longer secondary drying times. The TGA results of this lyophilization study showed some variability in mass loss from 18% w/w to 25% w/w, while one vial tested with the study employing the longer secondary drying time had a weight loss of 18%.

The DMSO amounts for the two lyophilization cycles are shown in table 5 below:

example 2: the full load confirmation study of the lyophilization process of table 1.

The aim of this study was to run full-load vials with the first batch of GMP compound of formula (1) to demonstrate that the purification cycle is safe, effective and robust. Bulk solution formulated with compound of formula (1) was filled into 1620 vials on four trays at a target fill volume of 1 mL. At the end of compounding, there are bright white and floating large foreign particles in the solution. Thermocouples were placed into 4 edge vials and 6 center vials. After loading is complete, the chamber is evacuated to 11-13PSIA to ensure that the chamber is properly sealed. The product was freeze-dried according to the process parameters in table 6.

Figure 37 provides the lyophilization cycle parameter results.

Figure 38 provides RGA data for the lyophilization parameters of table 6. Throughout primary drying, RGA detected DMSO in the chamber. After about 55 hours of primary drying, the signal approaches baseline levels. During secondary drying, RGA did not detect a second increase in DMSO levels.

A summary of the product temperatures at equilibrium is provided in table 7 below:

¹the product temperature indicates the temperature at the end of the run.

²Indicating the temperature of the product immediately prior to evacuation.

A summary of the product off temperatures is shown in table 8 below:

at the completion of the study, a 100% check was made for physical appearance. Each tray was reconstituted for 9 center vials and 9 edge vials. Turbidity tests were performed by combining 3 reconstituted vials per sample.

Fig. 39 shows a top view of a vial of lyophilized product. The product appeared to be a compact yellow block cake. The original fill height was 5mm and the product height was 4mm, with uniform shrinkage observed around the 1mm side. The top of the cake appeared matte with some areas glossy, while the sides and bottom appeared matte. The top of the cake was concave and its texture was cracked at the cake height. Upon inversion, the cake spreads and moves to the top of the vial. Upon shaking, the cake moved to the top of the vial and broke into pieces and powder. There was a minimal amount of residual material around the original fill level, as a thin yellow film.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. The reconstitution time for this study was reported in minutes due to the long reconstitution time. Table 9 provides the mean reconstitution time and turbidity results for this study.

The TGA analysis results are provided in table 40. The core product temperature was kept below the critical temperature of-4 ℃ until the interruption was reached. The product temperature of the edge thermocouple is slightly higher than the critical temperature during interruption; however, no sign of collapse or meltback was observed in the edge vials. After about 55 hours of primary drying, the product temperature reached steady state. The TGA test results showed a total weight loss of 17%, consistent with previous results.

Example 3: another lyophilization process for the compound of formula (1).

A series of vials containing a solution containing a compound of formula (1) were lyophilized using the specific cycling parameters listed in table 10 below.

After the lyophilization cycle is complete, the lyophilizer is backfilled with nitrogen and the vials are completely and automatically stoppered. The vials were aseptically transferred into isolators where each vial was automatically covered with a blue aluminum flip cap. The vials were visually inspected, followed by sampling for release testing, and labeling and packaging operations. The vial was kept at 2-8 ℃ until ready for use. Each vial is labeled with its contents.

Example 4: and (5) comparing and testing.

I. A lyophilized formulation prepared by the method disclosed herein:

bulk solutions containing the sodium salt of the compound of formula (1) were prepared in DSMO at four different concentrations and the resulting solutions (labeled a to D) were filled into lyophilization vials and lyophilized using the protocol described in example 1 above. The end of the primary drying (sublimation) phase was determined using Pirani and Baratron instruments. Figure 1 shows the gradual decrease in DMSO content over time during the primary and secondary drying stages.

After lyophilization, the lyophilized samples were analyzed for purity (% purity determined by HPLC), DMSO residual, and residual moisture. Samples were reconstituted by dissolving them in a non-aqueous solvent system as described in table 11 below, and the reconstituted formulations were analyzed for reconstitution time and appearance.

The analysis results are shown in table 12 below.

Results for four different concentrations, n ═ 1

LOQ is the limit of quantitation

Comparative formulation ii:

a bulk solution of the sodium salt of the compound of formula (1) at a concentration of 100mg/mL was lyophilized using the apparatus described in example 3 above but with a different temperature profile which did not include the first warming stage during freezing of the solution but which included freezing the formulation at a different freezing rate.

The properties of the comparative formulation prepared in this manner are shown in table 13 below.

Comparison of the results obtained from the formulations described in I and II

The results shown in step I above indicate that when an intermediate warming phase ("first warming phase") is included in the solution freezing process prior to primary drying according to the processes disclosed herein, the result is a lyophilized dry formulation that can be reconstituted below 20 minutes and in some cases below 15 minutes.

In contrast, the comparative formulations FP1, FP2, and FP3 described in II above, prepared by a procedure that omits an intermediate warming phase, took longer to reconstitute (over 30 minutes). The intermediate warming stage can have the effect of increasing the porosity of the lyophilized product and increasing the surface area available for contact with solvent molecules, thereby increasing the solubility of the formulation.

Comparison of drying time with example 4 in WO2013/033176

Example 4 in WO2013/033176 describes the lyophilization of a solution of the sodium salt of the compound of formula (1) using the cycling parameters shown in table 14 below.

In the process of the present disclosure, when the solution is initially frozen, an intermediate (first) warming phase is inserted between the two freezing phases, which is believed to result in a more porous structure from which DMSO can more easily sublime during the primary drying phase. Thus, a greater proportion of DMSO is removed in the primary drying stage, with the result that a much shorter secondary drying stage can be used.

Thus, in summary, the process of the present disclosure can reduce the time necessary to produce a lyophilized product with greatly enhanced dissolution characteristics.

Example 5: larger scale studies of 75mg/mL and 100mg/mL formulations A and B.

The results obtained in the experiment described in example 3 show that the lowest residual DMSO levels were obtained with formulation B, which was lyophilized from a bulk solution containing the active compound at a concentration of 75 mg/mL. Therefore, confirmatory studies were performed on 75mg/mL and 100mg/mL solutions of the sodium salt of the compound of formula (1) in DMSO. Lyophilization was performed on a 100 vial scale and multiple samples were analyzed. The protocol used is as described in example 3. The properties of the resulting lyophilized product are shown in table 15 below.

Reconstitution time does not include dissipation of bubbles (about 10 minutes). However, the reconstitution is performed manually, without the need for mechanical mixing devices.

Although not seen in this case, in some cases the solution may be slightly cloudy and/or slightly off-white to yellow.

The results in table 15 demonstrate that the process of the present disclosure can be used, for example, to prepare lyophilized formulations with reconstitution times shorter than 10 minutes (not including the time required to clear air bubbles), and that reconstitution can be performed manually without the need for a mechanical mixer.

Example 6: preparation of the sodium salt of the Compound of formula (1)

The sodium salt of the compound of formula (1) is prepared as described in US 7700567 (the contents of which are incorporated herein by reference) which comprises reacting 1s (wherein R is₁A carbamate protecting group) with phosphoramidite building block 1 d:

protected 2' -deoxyguanosine-linked CPG solid support 1s (where R is R) was prepared in the presence of 60% of 0.3M benzylthiotetrazole activator in acetonitrile₁Tertbutylphenoxyacetyl) with 2-2.5 equivalents of phenoxyacetyl decitabine phosphoramidite (1d, wherein R is₁Phenoxyacetyl) for 10 minutes. The CPG solid support containing the protected DpG di-nucleotide was applied with 20mL of 50mM K₂CO₃Treatment (in methanol) for 1 hour 20 minutes. The coupling product was oxidized to remove the protecting group and the resulting compound was washed, filtered and passed through a Gemini C18 preparative column (Phenomenex), 250X21.2mm, 10 μm and guard column (Phenomenex), 50X21.2mm, 10 μm

Explorer 100HPLC was used for purification using 50mM triethylammonium acetate (pH 7) in MilliQ water (mobile phase A) and 80% acetonitrile in MilliQ water (mobile phase B), using 2% to 20/25% mobile phase B in the column volume.

ESI-MS (-ve) of DpG dinucleotide 2 b:

wherein X⁺Triethylammonium (neutral compound C)₁₈H₂₄N₉O₁₀P has a calculated exact molecular weight of 557.14), showing M/z 556.1[ M-H ]]^-And 1113.1[2M-H]^-。

The sodium salt of a compound of formula (1), i.e. DpG dinucleotide 2b, wherein X⁺Sodium, obtained as follows: the triethylammonium salt was redissolved in 4mL of water, 0.2mL of 2M NaClO₄In solution. When 36mL of acetone was added, the dinucleotide precipitated out. The solution was held at-20 ℃ for several hours and centrifuged at 4000rpm for 20 minutes. The supernatant was discarded and the solid was washed with 30mL of acetone, followed by re-centrifugation at 4000rpm for 20 minutes. The precipitate dissolved in water and freeze-dried showed M/z 556.0[ M-H ]]^-。

Example 7: confirmation of lyophilization process for compounds disclosed herein.

The results of the lyophilization procedure shown in table 16 are clear reconstituted solutions, as opposed to turbid solutions obtained by other lyophilization procedures.

Example 8: 100mg of compound of formula (1) presented for injection per vial was evaluated by the process of objective and boundary studies.

To demonstrate the safety, effectiveness, and robustness of the lyophilization process of example 7, a series of studies were conducted in which the various steps of the process were adjusted to be at or above or below a target set point. This project was originally designed to consist of the following studies: one study used target process parameters, and the following four studies used a shelf temperature of target conditions of ± 3 ℃ in combination with a primary drying chamber pressure of target conditions of ± 5 microns and a secondary drying chamber pressure of ± 175 microns to demonstrate a proven acceptable range.

Deviations occurred during the initial study, where the chamber pressure in the secondary drying did not increase based on the target chamber pressure of 200 microns. Therefore, three additional studies were included to demonstrate the actual target parameters and the combination of high chamber pressure and high and low shelf temperatures in secondary drying. A summary of the conditions used in each study is included in table 17.

During the study G process, the system experienced a primary dry shelf temperature without evacuating the chamber to a high boundary chamber pressure of 25 microns; however, no effect on the product was observed as the product temperature remained frozen below the annealing temperature for the entire time.

No process data from study H was collected during freezing, annealing and refreezing. The data collected at the end of the secondary freezing and in the ramp-to-primary drying indicated that the freeze dryer was programmed to perform the freezing, annealing, secondary freezing, and evacuation steps. Thus, no effect on the purpose of this study was observed, as the high shelf temperature freezing process has been demonstrated to be acceptable in studies B and D.

All studies produced a slightly brittle cake which, when reconstituted with a diluent, formed a clear and colorless low turbidity solution. Low shelf temperature and low chamber pressure studies require the longest time to complete sublimation. The high shelf temperature study performed sublimation well within the indicated time while still maintaining the structure formed during freezing.

These objective and boundary studies successfully demonstrated a range of objective lyophilization processes: 3 ℃ around the target shelf temperature in each station, 5 microns around the target chamber pressure in primary drying and 175 microns in secondary drying.

For all studies, the compound of formula (1) was stored at 2 ℃ to 8 ℃ until use. At the time of use, the compound of formula (1) was weighed and dispensed into about 90% of the total volume of DMSO. The amount of compound of formula (1) was adjusted according to the assay (as such, free acid) reported in the certificate of analysis of each batch. The DMSO was mixed vigorously with a magnetic stir bar for about 2 hours until all compounds were dissolved. Once all compounds were dissolved, the solution was adjusted appropriately to a final concentration of 100mg/mL compound using additional DMSO, assuming a density of 1.164 g/mL. The solution was then filtered through a 0.2 μm filter.

The general processing procedure is provided below:

1. compounds of formula (1) were formulated and filtered according to the respective batch recording procedure.

2. A washed 6R I type vial (Schott part No. 1123261) was filled to a target fill volume of 1mL with a bulk solution of the compound for injection.

3. A West 20mm single vent 4432/50G B2-TR stopper (part number 19700033) was partially inserted into the vial.

4. Thermocouples were placed in the bottom center of 10 product vials, including 6 center vials and 4 edge vials.

5. The bottomless tray containing the product was placed on the shelf of the lyophilizer and the bottom of the tray was removed. Bulk trays (bulk tray) loaded with DMSO and spacers were placed on any shelf without product.

6. After loading the product, the chamber was evacuated to about 12PSIA to ensure good door sealing.

7. The lyophilization cycle was completed according to the schedule using the general parameters outlined in the following section. Data was recorded electronically every 5 minutes.

The target lyophilization cycle for the compound of formula (1) used for the comparative study was:

1. the shelves were controlled at a target set point of 20 ℃ until product was loaded onto the shelves. The temperature was held for 1 hour to allow all product samples to equilibrate at the target temperature.

2. The shelves were cooled at an average controlled rate of 30 ℃/hour to a target shelf set point of-45 ℃. The target shelf set point was maintained for 1 hour to allow all product to equilibrate and fully cure at the target temperature.

3. The shelves were ramped up to a target shelf temperature set point of 0 ℃ at an average controlled rate of 30 ℃/hour. The target shelf temperature was maintained at the set point for 2 hours to allow all product samples to anneal at the target temperature.

4. The shelves were cooled at an average controlled rate of 30 ℃/hour to a target shelf set point of-45 ℃. The target shelf set point was maintained for 2 hours to allow all product samples to equilibrate and fully cure at the target temperature.

5. The condenser was cooled to below-40 ℃ and the chamber was evacuated to the target pressure. The target shelf set point was held for an additional 4 hours to allow any unfrozen DMSO to evaporate.

6. The chamber pressure was controlled at a target set point to sublime the DMSO.

7. The shelves were ramped up to a target shelf temperature set point of-6 ℃ at an average controlled rate of 30 ℃/hour and controlled at the target shelf set point for 80.5 hours until all DMSO had sublimated.

8. The shelves were ramped up at an average controlled rate to a target shelf temperature setpoint of 40 ℃ and held at the target shelf setpoint to reduce the residual DMSO level.

9. The shelf was cooled to a target set point of 20 ℃ for unloading. The chamber pressure was raised to 14.7 ± 0.7PSIA by charging the chamber with filtered nitrogen NF. The vial was stoppered and unloaded.

Table 18 below provides a summary of the process parameters for the boundary study:

¹after 2 hours a vacuum was pulled to the set point listed at the end of the table and the pressure was maintained for the remainder of the process.

Diluents used in the following studies were:

study a: a targeted study with low chamber pressure was used.

The objective of this study was to reproduce the target lyophilization process to demonstrate the consistency of the boundary study with the target lyophilization parameters. The bulk solution was filled into approximately 400 vials on one tray at a target fill volume of 1 mL. Full load conditions were simulated with bulk trays with DMSO. Thermocouples were placed into 4 edge vials and 6 center vials. After loading is complete, the chamber is evacuated to 11-13PSIA to ensure that the chamber is properly sealed. The product was freeze dried according to the original target process parameters, which did not include the 200 micron chamber pressure set point in secondary drying.

During compounding, a white particle with a diameter of about 3-5mm was observed to float in solution. The particle is considered a foreign substance because it appears brighter than the drug substance, and the particle floats while most drug substances sink. Therefore, the study was continued without the particles being dissolved.

The lyophilization cycle parameter results are shown in fig. 5.

A summary of the product temperatures at equilibrium is provided in table 20 below:

¹the product temperature indicates the temperature at the end of the run.

²Indicating the temperature of the product immediately prior to evacuation.

A summary of the product off temperatures is shown in table 21 below:

at the completion of the study, a 100% check was made for physical appearance. The 9 vials were reconstituted. Turbidity tests were performed by combining 3 reconstituted vials per sample. DSC and TGA were performed on both vials. Fig. 6 shows a top view of a vial of lyophilized product. The physical appearance of the product vial showed a uniform, dense white cake with uniform shrinkage around the sides.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. The reconstitution time for this study was reported in minutes due to the long reconstitution time. Table 22 provides the mean reconstitution time for this study. Reconstitution was done with 1mL of diluent and it took about 20 minutes for the solution to be completely clear. The reconstitution time is consistent with or slightly shorter than previous studies with compounds of formula (1) for injection. Turbidity tests of all samples showed NTU values below 3, which is considered as a clear solution with turbidity not exceeding that of the diluent.

The TGA analysis results are provided in table 23 below.

DSC results are provided in figure 7 below. Figure 7 provides an overlay between DSC and TGA thermograms. The TGA results showed a mass loss of about 19% w/w. DSC showed that baseline shifts were associated with various weight loss events as determined by TGA, suggesting that changes in DSC were related to the escape of residual DMSO in the sample. For all vials monitored with thermocouples, the product temperature was kept below the critical temperature of-4 ℃ until the interruption was reached. After about 64 hours of primary drying, the product temperature reached steady state.

This study demonstrated that using the recommended target parameters in primary drying and low pressure in secondary drying, a refined product was obtained with consistent residual DMSO levels and low turbidity after reconstitution. This study represents the effect of using low pressure in the secondary drying and using the target primary drying to establish the difference between the two pressure set points.

Study B: high shelf temperature, high chamber pressure

The objective of this study was to conduct the first of four boundary studies to demonstrate that the target lyophilization process is safe, effective, and robust. The bulk solution was filled into approximately 400 vials on one tray at a target fill volume of 1 mL. Full load conditions were simulated with bulk trays with DMSO. Thermocouples were placed into 4 edge vials and 6 center vials. After loading is complete, the chamber is evacuated to 11-13PSIA to ensure that the chamber is properly sealed. The product was freeze dried according to the high shelf temperature, high chamber pressure (HH) process parameters. Thus, in secondary drying, the chamber pressure was maintained at 25 microns. RGA is connected to the lyophilizer at a sample port located at the top of the lyophilizer chamber.

During compounding, the addition of the compound of formula (1) is monitored. The material tends to cake and the larger lumps will not wet properly. This tendency shortens the dissolution time and causes the material to float. Another wet mass was observed to sink and adhere to the bottom of the container. These materials remained undissolved and were then filtered for further analysis.

The lyophilization cycle parameter results are shown in fig. 8.

A summary of the product temperatures at equilibrium is provided in table 24 below:

¹the product temperature indicates the temperature at the end of the run.

²Indicating the temperature of the product immediately prior to evacuation.

A summary of the product off temperatures is shown in table 25 below:

the RGA data is shown in fig. 9. RGA data showed that DMSO signal increased at the beginning of primary drying. After about 54 hours of primary drying, the DMSO level returned to the baseline level.

At the completion of the study, a 100% check was made for physical appearance. The 9 vials were reconstituted. Turbidity tests were performed by combining 3 reconstituted vials per sample. DSC and TGA were performed on both vials. Fig. 10 shows a top view of a vial of lyophilized product. The physical appearance of the product vial showed a uniform, dense white cake with uniform shrinkage around the sides.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. For this study, the reconstitution time is reported in minutes. Table 26 provides the mean reconstitution time for this study. Reconstitution was done with 1mL of diluent and it took about 18 minutes for the solution to clear. Turbidity tests on all samples showed significantly higher NTU values than the target study. Duplicate tests were performed and all three samples showed NTU values below 3. Previous tests have shown that artificially high turbidity results may occur if the combined samples are not well mixed after combining.

¹Additional vials were tested after all other studies were completed because the first group of vials had an inconsistent trend with the other studies.

The TGA analysis results are provided in table 27 below.

DSC results are provided in figure 11 below. Figure 11 provides an overlay between DSC and TGA thermograms. The TGA results showed a mass loss of about 17% to 18% w/w. DSC showed that baseline shifts were correlated with various weight loss events as determined by TGA, consistent with the objective study.

For all central thermocouples, the product temperature was kept below the critical temperature of-4 ℃ before reaching the interruption. The interruption temperature of the edge thermocouple is-4 ℃ to-3 ℃; however, no effect on the finished product was detected. The central thermocouple product temperatures were all in the range of-8 ℃ to-6 ℃. After about 54 hours of primary drying, the product temperature reached steady state.

This study demonstrates that high shelf temperature and high chamber pressure boundary conditions in primary drying and relatively low chamber pressure in secondary drying yield a similar final product as the target study.

Study C: low shelf temperature, high chamber pressure

This study was the second of four boundary studies to demonstrate that the target lyophilization process is safe, effective, and robust. The bulk solution was filled into approximately 400 vials on one tray at a target fill volume of 1 mL. Full load conditions were simulated with bulk trays with DMSO. Thermocouples were placed into 4 edge vials and 6 center vials. After loading is complete, the chamber is evacuated to 11-13PSIA to ensure that the chamber is properly sealed. The product is freeze dried according to the low shelf temperature, high chamber pressure (LH) process parameters. Thus, in secondary drying, the chamber pressure was maintained at 25 microns. RGA is connected to the lyophilizer at a sample port located at the top of the lyophilizer chamber.

During compounding, the compound of formula (1) was checked for large lumps. These large pieces were broken up before adding the compound to DMSO to aid in dissolution. Complete dissolution of the compound was complete in about 1 hour. This result suggests that a problem previously presented for obtaining clear and colorless solutions is the dissolution properties of the compounds and the possible low shear mixing with a magnetic stir bar.

The lyophilization cycle parameter results are shown in fig. 12.

A summary of the product temperatures at equilibrium is provided in table 28 below:

¹the product temperature indicates the temperature at the end of the run.

²Indicating the temperature of the product immediately prior to evacuation.

A summary of the product interrupt temperatures is shown in table 29 below:

the RGA data is shown in fig. 13. RGA data showed that DMSO signal increased at the beginning of primary drying. After about 64 hours of primary drying, the DMSO level returned to the baseline level.

At the completion of the study, a 100% check was made for physical appearance. The 9 vials were reconstituted. Turbidity tests were performed by combining 3 reconstituted vials per sample. DSC and TGA were performed on both vials. Fig. 14 shows a top view of a vial of lyophilized product. The physical appearance of the product vial showed a uniform, dense white cake with uniform shrinkage around the sides.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. For this study, the reconstitution time is reported in minutes. Table 30 provides the mean reconstitution time for this study. Reconstitution takes about 18 to 19 minutes to clear the solution. Turbidity tests of all samples showed NTU values below 3, which is considered as a clear solution with turbidity not exceeding that of the diluent.

The TGA analysis results are provided in table 31 below.

DSC results are provided in figure 15 below. Figure 15 provides an overlay between DSC and TGA thermograms. The TGA results showed a mass loss of about 19% w/w. DSC showed that baseline shifts were correlated with various weight loss events as determined by TGA, consistent with the objective study.

For all monitored vials, the product temperature was kept below the critical temperature of-4 ℃ until the interruption was reached. Based on the thermal analysis data, the central thermocouple product temperatures were all below the target range of-8 ℃ to-6 ℃. After about 76 hours of primary drying, the product temperature reached steady state.

This study demonstrates that low shelf temperature and high chamber pressure boundary conditions in primary drying and relatively low chamber pressure in secondary drying yield a similar final product as the target study.

Study D: high shelf temperature, low chamber pressure

This study was the third of four boundary studies to demonstrate that the target lyophilization process is safe, effective, and robust. The bulk solution was filled into approximately 400 vials on one tray at a target fill volume of 1 mL. Full load conditions were simulated with bulk trays with DMSO. Thermocouples were placed into 4 edge vials and 6 center vials. After loading is complete, the chamber is evacuated to 11-13PSIA to ensure that the chamber is properly sealed. The product was freeze dried according to the high shelf temperature, low chamber pressure (HL) process parameters. Thus, in secondary drying, the chamber pressure was maintained at 15 microns. RGA is connected to the lyophilizer at a sample port located at the top of the lyophilizer chamber.

During compounding, the compound of formula (1) was checked for large chunks and the chunks were broken into smaller pieces prior to addition to DMSO. Dissolution was complete in about 2.5 hours due to the formation of larger lumps during the addition of the compound to DMSO.

The lyophilization cycle parameter results are shown in fig. 16.

A summary of the product temperatures at equilibrium is provided in table 32 below:

¹the product temperature indicates the temperature at the end of the run.

²Indicating the temperature of the product immediately prior to evacuation.

A summary of the product interrupt temperatures is shown in table 33 below:

the RGA data is shown in fig. 17. RGA data showed that DMSO signal increased at the beginning of primary drying. After about 49 hours of primary drying, the DMSO level returned to the baseline level.

At the completion of the study, a 100% check was made for physical appearance. The 9 vials were reconstituted. Turbidity tests were performed by combining 3 reconstituted vials per sample. DSC and TGA were performed on both vials. Fig. 18 shows a top view of a vial of lyophilized product. The physical appearance of the product vial showed a uniform, dense white cake with uniform shrinkage around the sides.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. For this study, the reconstitution time is reported in minutes. Table 34 provides the mean reconstitution time for this study. Reconstitution takes about 16 to 18 minutes to clear the solution. Turbidity tests of all samples showed NTU values below 3, which is considered as a clear solution with turbidity not exceeding that of the diluent.

The TGA analysis results are provided in table 35 below.

DSC results are provided in figure 19 below. Figure 19 provides an overlay between DSC and TGA thermograms. The TGA results showed a mass loss of about 17% w/w. DSC showed that baseline shifts were correlated with various weight loss events as determined by TGA, consistent with the objective study.

For all monitored vials, the product temperature was kept below the critical temperature of-4 ℃ until the interruption was reached. The interruption temperature of the edge thermocouple is-3 ℃ to-1 ℃; however, no effect on the finished product was detected. Based on the thermal analysis data, the central thermocouple product temperatures were all at the warmer end of the recommended target range of-8 ℃ to-6 ℃. After about 51 hours of primary drying, the product temperature reached steady state.

This study demonstrated that low shelf temperature and high chamber pressure boundary conditions and chamber pressures below 25 microns in secondary drying achieved a similar final product as the target study.

Study E: low shelf temperature, low chamber pressure

This study was the fourth of four boundary studies to demonstrate that the target lyophilization process is safe, effective, and robust. The bulk solution was filled into approximately 400 vials on one tray at a target fill volume of 1 mL. Full load conditions were simulated with bulk trays with DMSO. Thermocouples were placed into 4 edge vials and 6 center vials. After loading is complete, the chamber is evacuated to 11-13PSIA to ensure that the chamber is properly sealed. The product was freeze dried according to the low shelf temperature, low chamber pressure (LL) process parameters. Thus, in secondary drying, the chamber pressure was maintained at 15 microns.

During compounding, one batch of the compound of formula (1) took about seven hours to reach complete dissolution, while another batch of the compound of formula (1) took slightly less than 2.5 hours. This dissolution rate was attributed to the low volume formulated for the first batch, and the mixing achieved in the vessel with the magnetic stir bar, as the first batch was previously soluble in as little as 1 hour.

The lyophilization cycle parameter results are shown in fig. 20.

A summary of the product temperatures at equilibrium is provided in table 36 below:

¹the product temperature indicates the temperature at the end of the run.

²Indicating the temperature of the product immediately prior to evacuation.

A summary of the product off temperatures is shown in table 37 below:

at the completion of the study, a 100% check was made for physical appearance. The 9 vials were reconstituted. Turbidity tests were performed by combining 3 reconstituted vials per sample. DSC and TGA were performed on two vials per sub-batch. Panel a in fig. 21 shows a top view of a vial of lyophilized product from a first batch of compound of formula (1) that is difficult to dissolve, while panel B in fig. 21 shows a top view of a vial of lyophilized product from a second batch that dissolves faster than the first batch of compound of formula (1). The physical appearance of the product vial showed a uniform, dense white cake with uniform shrinkage around the sides.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. For this study, the reconstitution time is reported in minutes. Table 38 provides the mean reconstitution time for this study. For both sub-batches, reconstitution took 17 minutes. Turbidity tests of all samples showed NTU values below 3, which is considered as a clear solution with turbidity not exceeding that of the diluent.

The TGA analysis results are provided in table 39 below.

DSC results are provided in figure 22 below. Figure 22 provides an overlay between DSC and TGA thermograms. The TGA results show greater variability in mass loss for the first batch, but the second batch is consistent, being about 19% w/w. This variability may be due to lower shelf temperatures and lower chamber pressures, as these conditions reduce the rate of DMSO desorption. No effect on the product was observed, as the residual DMSO level was still within acceptable levels for the compound of formula (1) for injection. DSC showed that baseline shifts correlated with various weight loss events as determined by TGA, which shifts were consistent with the target study results.

For all monitored vials, the product temperature was kept below the critical temperature of-4 ℃ until the interruption was reached. According to the thermal analysis data, the product temperatures were all below the recommended target range of-8 ℃ to-6 ℃. After about 80.5 hours of primary drying, the product temperature approaches steady state.

This study demonstrated that low shelf temperature and low chamber pressure boundary conditions and chamber pressures in secondary drying slightly below 25 microns achieved a similar final product as the target study.

Study F: study of the object

The objective of this study was to repeat the target lyophilization process and demonstrate that the target lyophilization process is safe, effective, and robust. The bulk solution was filled into approximately 175 vials on one tray at a target fill volume of 1 mL. Full load conditions were simulated with bulk trays with DMSO. Thermocouples were placed into 4 edge vials and 6 center vials. After loading is complete, the chamber is evacuated to 11-13PSIA to ensure that the chamber is properly sealed. The product was freeze dried according to the target process parameters in table 18.

During compounding, batch 1 of the compound of formula (1) took about 30 minutes to reach complete dissolution, while batch 2 took about 1.5 hours.

The lyophilization cycle parameter results are shown in fig. 23.

A summary of the product temperatures at equilibrium is provided in table 40 below:

¹the product temperature indicates the temperature at the end of the run.

²Indicating the temperature of the product immediately prior to evacuation.

A summary of the product off temperatures is shown in table 41 below:

at the completion of the study, a 100% check was made for physical appearance. The 9 vials were reconstituted. Turbidity tests were performed by combining 3 reconstituted vials per sample. DSC and TGA were performed on two vials per sub-batch. Panel a in fig. 24 shows a top view of vials of lyophilized product from a first batch of compound of formula (1), while panel B in fig. 24 shows a top view of vials of lyophilized product from a second batch. The physical appearance of the product vial showed a uniform, dense white cake with uniform shrinkage around the sides.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. For this study, the reconstitution time is reported in minutes. Table 42 provides the mean reconstitution time for this study. Reconstitution took about 17-18 minutes to clear the solution. The reconstitution time is consistent with or slightly shorter than previous studies with compound injection of formula (1). The turbidity test showed that the NTU value for batch 1 was lower than 1NTU and the NTU value for batch 2 was about 10NTU, which is consistent with the previous values for the two batches of API.

The TGA analysis results are provided in table 43 below.

DSC results are provided in figure 25 below. Figure 25 provides an overlay between DSC and TGA thermograms. The TGA results showed a mass loss of about 19% w/w. DSC showed that baseline shifts were associated with various weight loss events as determined by TGA, suggesting that changes in DSC were related to the escape of residual DMSO in the sample.

After about 40 hours of primary drying, the product reached a steady state.

This study demonstrated that the recommended target parameters resulted in a product with consistent residual DMSO levels and low turbidity after reconstitution.

Study G: low shelf temperature, high chamber pressure

The purpose of this study was to conduct an LH margin study to demonstrate that the lyophilization process is safe, effective, and robust. The bulk solution was filled into 140 vials on one tray at a target fill volume of 1 mL. The remaining vials and bulk trays were filled with DMSO to simulate full load conditions. Thermocouples were placed in 4 edge vials and 6 center vials; however, one thermocouple in the central vial was not recorded throughout the lyophilization process. After loading is complete, the chamber is evacuated to 11-13PSIA to ensure that the chamber is properly sealed. The product was freeze dried according to the LH process parameters in table 18.

During compounding, the compound of formula (1) was checked for large lumps. The batch of compound of formula (1) used in this study took about 3.3 hours to reach dissolution.

The lyophilization cycle parameter results are shown in fig. 26. Extended maintenance occurs at-9 ℃ using a near atmospheric system. No effect on the study due to this retention was observed.

A summary of the product temperatures at equilibrium is provided in table 44 below:

¹the product temperature indicates the temperature at the end of the run.

²Indicating the temperature of the product immediately prior to evacuation.

A summary of the product off temperatures is shown in table 45 below:

at the completion of the study, a 100% check was made for physical appearance. The 9 vials were reconstituted. Turbidity tests were performed by combining 3 reconstituted vials per sample. DSC and TGA were performed on one vial.

Fig. 27 shows a top view of a vial of lyophilized product. The physical appearance of the product vial showed a uniform, dense white cake with uniform shrinkage around the sides.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. For this study, the reconstitution time is reported in minutes. Table 46 provides the mean reconstitution time for this study. Reconstitution took slightly less than 17 minutes to clear the solution. Turbidity tests on all samples showed NTU values of about 15-20 NTU, which is consistent with previous results for this batch of compound of formula (1).

The TGA analysis results are provided in table 47 below.

DSC results are provided in figure 28 below. Figure 28 provides an overlay between DSC and TGA thermograms. The TGA results showed a mass loss of about 17% w/w, slightly lower than that of the target study. DSC showed that baseline shifts correlated with various weight loss events as determined by TGA, which shifts were consistent with the target study results.

For all monitored vials, the product temperature was kept below the critical temperature of-4 ℃ until the interruption was reached. According to the thermal analysis data, all product temperatures were below the target range of-8 ℃ to-6 ℃. After about 75 hours of primary drying, the product temperature reached steady state. After about 55 hours of primary drying, the hastings meter was returned to match the chamber pressure.

This study demonstrated that low shelf temperature and high chamber pressure boundary conditions yielded a finished product similar to the target study.

Study H: high shelf temperature, high chamber pressure

The objective of this study was to conduct HH boundary studies to demonstrate that the target lyophilization process is efficient and robust. The bulk solution was filled into 135 vials on one tray at a target fill volume of 1 mL. The remaining vials and bulk trays were filled with DMSO to simulate full load conditions. Thermocouples were placed in 3 edge vials and 6 center vials, but one edge thermocouple did not record any data. After loading is complete, the chamber is evacuated to 11-13PSIA to ensure that the chamber is properly sealed. The product was freeze-dried according to HH process parameters in table 18; however, no data was collected during the freezing and annealing steps. RGA was connected to the lyophilizer at a sample port located at the back of the lyophilizer chamber.

During compounding, the compound of formula (1) was checked for large lumps. The batch of compound of formula (1) used in this study took about 116 minutes to reach complete dissolution. The initial bulk solution was stored at 2 ℃ to 8 ℃ rather than controlled at room temperature and inadvertently frozen. A second bulk solution was prepared with a dissolution time of 46 minutes. The second bulk solution was filled into vials.

The lyophilization cycle parameter results are shown in fig. 29. Note that: TC-2 was excluded from further data analysis.

A summary of the product temperatures at equilibrium is provided in table 48 below:

¹the product temperature indicates the temperature at the end of the run.

The RGA data is shown in fig. 30. RGA data showed that DMSO signal increased at the beginning of primary drying. After about 61 hours of primary drying, the DMSO level returned to baseline.

A summary of the product interrupt temperatures is shown in table 49 below:

at the completion of the study, a 100% check was made for physical appearance. The 9 vials were reconstituted. Turbidity tests were performed by combining 3 reconstituted vials per sample. DSC and TGA were performed on one vial.

Fig. 31 shows a top view of a vial of lyophilized product. The physical appearance of the product vial showed a uniform, dense white cake with uniform shrinkage around the sides.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. The reconstitution time for this study was reported in minutes due to the long reconstitution time. Table 50 provides the mean reconstitution time for this study. Reconstitution took about 17 minutes to completely clear the solution, which was slightly faster than previous results. Turbidity tests on all samples showed NTU values of 3 to 32 NTU. These values have slightly higher variability than previous studies, but still within the same range consistent with previous results for this batch of the compound of formula (1).

The TGA analysis results are provided in table 51 below.

DSC results are provided in figure 32 below. Figure 32 provides an overlay between DSC and TGA thermograms. The TGA results showed a mass loss of about 17% w/w, slightly lower than that of the target study. DSC showed that baseline shifts correlated with various weight loss events as determined by TGA, which shifts were consistent with the target study results.

For all central thermocouples, the product temperature was kept below the critical temperature of-4 ℃ before reaching the interruption. The thermocouple product temperatures were all in the range of-8 ℃ to-6 ℃. After about 42 hours of primary drying, the product temperature reached steady state.

This study demonstrated that high shelf temperature and high chamber pressure boundary conditions yielded a similar final product as the target study.

As described above, data during freezing was not collected for the high shelf temperature, high chamber pressure study. Thus, the study was repeated, and the results are provided below.

The cycling parameters used in repeating the study are shown in table 52.

The lyophilization cycle parameters are also provided in fig. 33.

A summary of the product temperatures at equilibrium is provided in table 53 below:

table 54 provides a summary of the sublimations of this repeat study:

¹indicating the time the Hastings meter reading reaches steady state at chamber pressure during primary drying.

At the completion of the study, a 100% check was made for physical appearance. The 9 vials were reconstituted. Turbidity tests were performed by combining 3 reconstituted vials per sample. DSC and TGA were performed on 1 vial.

Fig. 34 provides a side view of the lyophilized product. Figure 35 provides a close-up top view of the product depicted in figure 34. The cake appeared dense with a uniform white color. The original fill height was 4-5mm and the product height was 4mm, with a uniform shrinkage of 1mm observed around the cake sides. The top, bottom and sides of the cake appeared glossy, with some matte bottom.

The top of the patty was textured and concave with cracks throughout the height of the patty. Upon inversion and shaking, the cake pieces moved to the top of the vial and broke further. Residual material was observed as a white film around the sides and bottom of the vial where the cake was originally located.

Reconstitution was performed by extruding 1mL of diluent into each vial using a vial adapter or syringe and allowing the vial to stand until clear. All samples yielded clear and colorless solutions. The reconstitution time for this study was reported in minutes due to the long reconstitution time. Table 55 provides the mean reconstitution time for this study. Reconstitution took about 12.5 minutes to completely clarify the solution. Turbidity tests on all samples showed NTU values less than or equal to 5NTU, which is in the same range as the previous results for this batch of compound of formula (1).

The TGA analysis results are provided in table 56 below.

DSC results are provided in figure 36 below. Figure 36 provides an overlay between DSC and TGA thermograms. The TGA results showed a mass loss of about 17% w/w, slightly lower than that of the target study, but consistent with the previous HH study. DSC showed that baseline shifts were correlated with various weight loss events as determined by TGA, consistent with the target study results.

For all central thermocouples, the product temperature was kept below the critical temperature of-4 ℃ before reaching the interruption. The interrupt temperature of the edge thermocouple is-3 ℃; however, no effect on the finished product was detected. The thermocouple product temperatures in the central vial were all slightly above the range of-8 ℃ to-6 ℃. After about 42 hours of primary drying, the product temperature reached steady state.

This study demonstrated that high shelf temperature and high chamber pressure boundary conditions yielded a similar final product as the target study.

Table 57 below provides a summary of the product temperature and TGA mass loss studies of all the foregoing studies:

table 58 below provides a summary of the sublimations for all the foregoing studies:

example 9: guadecitabine lyophilization cycle parameters.

Up to 30,000 vials of the compound of formula (1) were lyophilized using the procedure provided in table 59 below. All assay test results, including but not limited to assay, related substances, residual DMSO content, and reconstitution time, met the acceptance criteria.

Results from two example batches of different sizes were compared and the comparison is provided in table 60 below.

Example 10: impurities in the lyophilized drug product.

The batches of lyophilized product were analyzed to determine the purity of the product, type and amount of impurities, water content (mg/vial) and DMSO content (mg/vial). Impurities detected in the lyophilized product batch included the following compounds:

table 60 and table 61 show the content of the 7 batches of lyophilized drug product disclosed herein.

Detailed description of the preferred embodiments

The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

Embodiment 1. a method of preparing a lyophilized pharmaceutical composition comprising administering a compound of formula (1):

or a pharmaceutically acceptable salt thereof, in a solvent comprising dimethyl sulfoxide (DMSO) to form a solution, wherein the solvent is subsequently removed by a freeze-drying process to yield a lyophilized product, wherein the freeze-drying process comprises: (i) a first freezing stage wherein the solution is frozen by reducing its temperature to about-45 ℃; (ii) a first annealing stage in which the temperature of the frozen solution is raised to about 0 ℃, wherein a temperature of about 0 ℃ keeps the solution frozen; (iii) a second freezing stage wherein the temperature of the solution is reduced to a temperature of about-45 ℃; (iv) a primary drying stage, wherein the temperature of the solution is raised to about-6 ℃, wherein the primary drying stage comprises a sublimation step in which DMSO is removed by sublimation from the solution in a frozen state under reduced pressure to give a partially dried product; and (v) a secondary drying stage, wherein the temperature of the solution is raised to about 40 ℃, wherein in the secondary drying stage DMSO is removed by evaporation from the partially dried product in a non-frozen state under reduced pressure to give a lyophilized product.

Embodiment 2. the method of embodiment 1, wherein the compound of formula (1) is in the form of a sodium salt.

Embodiment 3. the method of any one of embodiments 1-2, wherein the solvent is non-aqueous.

Embodiment 4. the method of any of embodiments 1-3, wherein the lyophilized pharmaceutical composition has a dissolution time of no more than about 20 minutes in a non-aqueous solvent containing 65% (v/v) propylene glycol, 25% (v/v) glycerol, and 10% (v/v) ethanol at ambient temperature and without mechanical agitation.

Embodiment 5. the method of any of embodiments 1-4, wherein an amount of the lyophilized pharmaceutical composition obtained from 1 gram of the solution has a residual DMSO content of no more than about 20 mg.

Embodiment 6. the method of any of embodiments 1-5, wherein any residual DMSO present in the lyophilized pharmaceutical composition is in an amount equivalent to no more than 35mg/100mg equivalent of the free base of the compound of formula (1).

Embodiment 7. the method of any of embodiments 1-6, further comprising packaging the lyophilized drug in a sealed drug container.

Embodiment 8 the method of any one of embodiments 1-7, further comprising dissolving the lyophilized pharmaceutical composition in a solvent to form an injectable liquid composition.

Embodiment 9. the method of embodiment 8, wherein the solvent is a non-aqueous solvent.

Embodiment 10. the method of any of embodiments 1-9, wherein the solution further comprises a co-solvent.

Embodiment 11 the method of any one of embodiments 1-10, further comprising reconstituting the lyophilized pharmaceutical composition in a pharmaceutically acceptable solvent to obtain a liquid formulation comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof.

Embodiment 12. the method of any one of embodiments 1-11, wherein the reduced pressure in the primary drying stage is about 5 μ Bar to about 40 μ Bar.

Embodiment 13. the method of any one of embodiments 1 to 12, wherein the pressure in the secondary drying stage is about 5 μ Bar to about 40 μ Bar.

Embodiment 14. the method of any one of embodiments 1 to 13, wherein the pressure in the first freezing stage is about 750 μ Bar to about 850 μ Bar.

Embodiment 15. the method of any one of embodiments 1 to 14, wherein the pressure in the annealing stage is about 750 μ Bar to about 850 μ Bar.

Embodiment 16. a pharmaceutical composition prepared by a process comprising the steps of: reacting a compound of formula (1):

or a pharmaceutically acceptable salt thereof, in a solvent comprising dimethyl sulfoxide (DMSO) to form a solution, wherein the solvent is subsequently removed by a freeze-drying process to yield a lyophilized product, wherein the freeze-drying process comprises: (i) a first freezing stage wherein the solution is frozen by reducing its temperature to about-45 ℃; (ii) a first annealing stage in which the temperature of the frozen solution is raised to about 0 ℃, wherein a temperature of about 0 ℃ keeps the solution frozen; (iii) a second freezing stage wherein the temperature of the solution is reduced to a temperature of about-45 ℃; (iv) a primary drying stage, wherein the temperature of the solution is raised to about-6 ℃, wherein the primary drying stage comprises a sublimation step in which DMSO is removed by sublimation from the solution in a frozen state under reduced pressure to give a partially dried product; and (v) a secondary drying stage, wherein the temperature of the solution is raised to about 40 ℃, wherein in the secondary drying stage DMSO is removed by evaporation from the partially dried product in a non-frozen state under reduced pressure to give a lyophilized product.

Embodiment 17. the pharmaceutical composition of embodiment 16, wherein the compound of formula (1) is in the form of a sodium salt.

Embodiment 18. the pharmaceutical composition according to any one of embodiments 16-17, wherein the solvent is non-aqueous.

Embodiment 19 the pharmaceutical composition of any one of embodiments 16-18, wherein the lyophilized pharmaceutical composition has a dissolution time of no more than about 20 minutes in a non-aqueous solvent containing 65% (v/v) propylene glycol, 25% (v/v) glycerol, and 10% (v/v) ethanol at ambient temperature and without mechanical agitation.

Embodiment 20 the pharmaceutical composition of any one of embodiments 16-19, wherein an amount of lyophilized pharmaceutical composition obtained from 1 gram of the solution has a residual DMSO content of no more than about 20 mg.

Embodiment 21. the pharmaceutical composition according to any one of embodiments 16-20, wherein any residual DMSO present in the lyophilized pharmaceutical composition is in an amount equivalent to no more than 35mg/100mg equivalent of the free base of the compound of formula (1).

Embodiment 22. the pharmaceutical composition of any one of embodiments 16-21, the method further comprising packaging the lyophilized drug in a sealed drug container.

Embodiment 23. the pharmaceutical composition according to any one of embodiments 16-22, the method further comprising dissolving the lyophilized pharmaceutical composition in a solvent to form an injectable liquid composition.

Embodiment 24. the pharmaceutical composition of embodiment 23, wherein the solvent is a non-aqueous solvent.

Embodiment 25. the pharmaceutical composition according to any one of embodiments 16-24, wherein the solution further comprises a co-solvent.

Embodiment 26. the pharmaceutical composition according to any one of embodiments 16-25, the method further comprising reconstituting the lyophilized pharmaceutical composition in a pharmaceutically acceptable solvent to obtain a liquid formulation comprising a compound of formula (1) or a pharmaceutically acceptable salt thereof.

Embodiment 27. the pharmaceutical composition according to any one of embodiments 16-26, wherein the reduced pressure in the primary drying stage is about 5 μ Bar to about 40 μ Bar.

Embodiment 28. the method of any one of embodiments 16-27, wherein the pressure in the secondary drying stage is about 5 μ Bar to about 40 μ Bar.

Embodiment 29. the method of any one of embodiments 16-28, wherein the pressure in the first freezing stage is about 750 μ Bar to about 850 μ Bar.

Embodiment 30. the method of any one of embodiments 16-29, wherein the pressure in the annealing stage is about 750 μ Bar to about 850 μ Bar.

Embodiment 31 a composition comprising:

a) a compound of the formula:

or a pharmaceutically acceptable salt thereof,

wherein the composition comprises at least 95% of a compound of formula (1); and

b) a nucleotide-based compound which is not a compound of formula (1).

Embodiment 32 the composition of embodiment 31, wherein the nucleotide-based compound is a compound of formula (2):

or a pharmaceutically acceptable salt thereof,

wherein:

R¹is heteroaryl or urea, each of which is independently substituted or unsubstituted;

each R²And R³Independently substituted or unsubstituted alkyl; or hydrogen; and is

R⁴Is hydrogen or acyl, each of which is independently substituted or unsubstituted.

Embodiment 33. the composition of embodiment 32, wherein R¹Is a substituted urea.

Embodiment 34. the composition of embodiment 32, wherein R¹Is heteroaryl.

Embodiment 35. the composition of embodiments 32 or 34, wherein R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

Embodiment 36. the composition of any of embodiments 32-35, wherein each R²And R³Is substituted alkyl or hydrogen.

Embodiment 37. the composition of any of embodiments 32-36, wherein R²Is H and R³Is methyl substituted by methoxy.

Embodiment 38. the composition of any of embodiments 32-37, wherein R⁴Is hydrogen.

Embodiment 39. the composition of any of embodiments 32-37, wherein R⁴Is an acyl group.

Embodiment 40. the composition of embodiment 32, wherein the compound of formula (2) is

Embodiment 41. the composition of embodiment 32, wherein the compound of formula (2) is

Embodiment 42. the composition of embodiment 32, wherein the compound of formula (2) is

Embodiment 43. the composition of embodiment 32, wherein the compound of formula (2) is

Embodiment 44. the composition of embodiment 31, wherein the nucleotide-based compound is a compound of formula (3):

or a pharmaceutically acceptable salt thereof, wherein R¹Is heteroaryl or urea, each of which is independently substituted or unsubstituted.

Embodiment 45. the composition of embodiment 44, wherein R¹Is heteroaryl.

Embodiment 46. the composition of embodiment 44 or 45, wherein R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

Embodiment 47. the composition of embodiment 44, wherein R¹Is a substituted urea.

Embodiment 48. the composition of embodiment 44, wherein the compound of formula (3) is

Embodiment 49. the composition of embodiment 44, wherein the compound of formula (3) is

Embodiment 50 the composition of embodiment 31, wherein the nucleotide-based compound is a compound of formula (4):

or a pharmaceutically acceptable salt thereof, wherein R¹Is a substituted or unsubstituted heteroaryl; and R is⁵Is hydroxyl or nucleotide.

Embodiment 51. the composition of embodiment 50, wherein R¹Is a substituted heteroaryl group.

Embodiment 52. the composition of embodiments 50 or 51, wherein R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

Embodiment 53. the composition of embodiment 50 or 51, wherein R¹Is 2-amino-9 lambda²-purin-6 (1H) -one.

Embodiment 54. the composition of any of embodiments 50 to 53, wherein R⁵Is a hydroxyl group.

Embodiment 55. the composition of any of embodiments 50 to 53, wherein R⁵Are nucleotides.

Embodiment 56. the composition of any one of embodiments 50-53 and 55, wherein the nucleotide has the formula:

embodiment 57 the composition of embodiment 50, wherein the compound of formula (4) is

Embodiment 58. the composition of embodiment 50, wherein the compound of formula (4) is

Embodiment 58a. a pharmaceutical composition comprising, in a unit dosage form:

a) a compound of formula (1):

or a pharmaceutically acceptable salt thereof;

b) a nucleotide-based compound which is not a compound of formula (1); and

c) a pharmaceutically acceptable excipient.

Embodiment 59. the pharmaceutical composition of embodiment 58a, wherein the nucleotide-based compound is a compound of formula (2):

or a pharmaceutically acceptable salt thereof, wherein: r¹Is heteroaryl or urea, each of which is independently substituted or unsubstituted; each R²And R³Independently substituted or unsubstituted alkyl; or hydrogen; and R is⁴Is hydrogen or acyl, each of which is independently substituted or unsubstituted.

Embodiment 60 the pharmaceutical composition of embodiment 59, wherein R¹Is a substituted urea.

Embodiment 61 the pharmaceutical composition of embodiment 59, wherein R¹Is heteroaryl.

Embodiment 62 according to embodiment 59 or 61The pharmaceutical composition of (1), wherein R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

Embodiment 63. the pharmaceutical composition of any one of embodiments 59-62, wherein each R²And R³Is substituted alkyl or hydrogen.

Embodiment 64. the pharmaceutical composition according to any one of embodiments 59-63, wherein R²Is H and R³Is methyl substituted by methoxy.

Embodiment 65. the pharmaceutical composition according to any one of embodiments 59-64, wherein R⁴Is hydrogen.

Embodiment 66. the pharmaceutical composition according to any one of embodiments 59-64, wherein R⁴Is an acyl group.

Embodiment 67. the pharmaceutical composition of embodiment 59, wherein the compound of formula (2) is

Embodiment 68. the pharmaceutical composition of embodiment 59, wherein the compound of formula (2) is

Embodiment 69 the pharmaceutical composition of embodiment 59, wherein the compound of formula (2) is

Embodiment 70 the pharmaceutical composition of embodiment 59, wherein the compound of formula (2) is

Embodiment 71. the pharmaceutical composition of embodiment 58a, wherein the nucleotide-based compound is a compound of formula (3):

or a pharmaceutically acceptable salt thereof, wherein R¹Is heteroaryl or urea, each of which is independently substituted or unsubstituted.

Embodiment 72 the pharmaceutical composition of embodiment 71, wherein R¹Is heteroaryl.

Embodiment 73. the pharmaceutical composition of embodiment 71 or 72, wherein R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

Embodiment 74 the pharmaceutical composition of embodiment 71, wherein R¹Is a substituted urea.

Embodiment 75. the pharmaceutical composition of embodiment 71, wherein the compound of formula (3) is

Embodiment 76. the pharmaceutical composition of embodiment 71, wherein the compound of formula (3) is

Embodiment 77 the pharmaceutical composition of embodiment 58a, wherein the nucleotide-based compound is a compound of formula (4):

or a pharmaceutically acceptable salt thereof, wherein R¹Is a substituted or unsubstituted heteroaryl; and R is⁵Is hydroxyl or nucleotide.

Embodiment 78 the pharmaceutical composition of embodiment 77, wherein R¹Is a substituted heteroaryl group.

Embodiment 79. the pharmaceutical composition of embodiment 77 or 78, wherein R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

Embodiment 80. the pharmaceutical composition of embodiment 77 or 78, wherein R¹Is 2-amino-9 lambda²-purin-6 (1H) -one.

Embodiment 81. the pharmaceutical composition according to any one of embodiments 77-80, wherein R⁵Is a hydroxyl group.

Embodiment 82. the pharmaceutical composition of any one of embodiments 77-80, wherein R⁵Are nucleotides.

Embodiment 83. the pharmaceutical composition of any one of embodiments 77-80 or 82, wherein the nucleotide has the formula:

embodiment 84. the pharmaceutical composition of embodiment 77, wherein the compound of formula (4) is

Embodiment 85. the pharmaceutical composition of embodiment 77, wherein the compound of formula (4) is

Embodiment 85a. the pharmaceutical composition according to any one of embodiments 58a-85, wherein the compound of formula (1) and the nucleotide-based compound are present in a ratio of about 20,000: about 1, about 19,000: about 1, about 18,000: about 1, about 17,000: about 1, about 16,000: about 1, about 15,000: about 1, about 14,000: about 1, about 13,000: about 1, about 12,000: about 1, about 11,000: about 1, about 10,000: about 1, about 9,900: about 1, about 9,800: about 1, about 9,700: about 1, about 9,600: about 1, about 9,500: about 1, about 9,400: about 1, about 9,300: about 1, about 9,200: about 1, about 9,100: about 1, about 9,000: about 1, about 8,900: about 1, about 8,800: about 1, about 8,8: about 1, about 8,300: about 1, about 8: about 1, about 8,800: about 1, about 1,200: about 1, about 8,800, about 1, about 7,600: about 1, about 7,500: about 1, about 7,400: about 1, about 7,300: about 1, about 7,200: about 1, about 7,100: about 1, about 7,000: about 1, about 6,900: about 1, about 6,800: about 1, about 6,700: about 1, about 6,600: about 1, about 6,500: about 1, about 6,400: about 1, about 6,300: about 1, about 6,200: about 1, about 6,100: about 1, about 6,000: about 1, about 5,900: about 1, about 5,800: about 1, about 5,700: about 1, about 5,600: about 1, about 5,500: about 1, about 5,400: about 1, about 5,300: about 1, about 5,200: about 1, about 5,100: about 1, about 5,000: about 1, about 4,500: about 1, about 3: about 1,1, about 3: about 1,400: about 1, about 3,400: about 1, about 1,200: about 1, about 1,1, about 3: about 1,500: about 1, about 1,1, about 3: about 4: about 1, about 1,400: about 1, about 1,200: about 1, about 3,500: about 1, about 1,400: about 1, about 3,400: about 1, about 1,200: about 1, about 1,2,2,2,2, about 1, about 3,300: about 1, about 3,200: about 1, about 3,100: about 1, about 3,000: about 1, about 2,900: about 1, about 2,800: about 1, about 2,700: about 1, about 2,600: about 1, about 2,500: about 1, about 2,400: about 1, about 2,300: about 1, about 2,200: about 1, about 2,100: about 1, about 2,000: about 1, about 1,900: about 1, about 1,800: about 1, about 1,700: about 1, about 1,600: about 1, about 1,500: about 1, about 1,400: about 1, about 1,300: about 1, about 1,200: about 1, about 1,100: about 1, about 1,000: about 1, about 990: about 1, about 980: about 1, about 950: about 1, about 1: 1, about 1,100: about 1, about 1: 90: about 1, about 1: 80: about 1, about 1: about 1, about 1: 80: about 1, about 1: 95: about 1, about 1: about 1, about 1: about 1, about 65: about 1, about 60: about 1, about 55: about 1, about 50: about 1, about 45: about 1, about 40: about 1, about 35: about 1, about 30: about 1, about 25: about 1, about 20: about 1, about 19: about 1, about 18: about 1, about 17: about 1, about 16: about 1, about 15: about 1, about 14: about 1, about 13: about 1, about 12: about 1, about 11: about 1, or about 10: about 1.

The pharmaceutical composition of any one of embodiments 58a-85, wherein the nucleotide-based compound is about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 1.9%, about 2%, about 3.1%, about 3.9%, about 3.3%, about 3.4%, about 3.6%, about 3.7%, about 3.9%, about 3.3%, about 3%, about 3.3%, about 3%, about 3.4%, about 3% by mass, About 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the total amount of the composition.

Embodiment 86. compounds of the formula:

or a pharmaceutically acceptable salt thereof, wherein: r¹Is heteroaryl or urea, each of which is independently substituted or unsubstituted; each R²And R³Independently substituted or unsubstituted alkyl; or hydrogen; and R is⁴Is hydrogen or acyl, each of which is independently substituted or unsubstituted, wherein the compound is not a compound of formula (1).

Embodiment 87. A compound according to embodiment 86, wherein R¹Is a substituted urea.

Embodiment 88. Compounds of embodiment 86 wherein R¹Is heteroaryl.

Embodiment 89. the compound of embodiment 86 or 88, wherein R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

Embodiment 90. the compound of any one of embodiments 86 to 89, wherein each R²And R³Is substituted alkyl or hydrogen.

Embodiment 91. Compounds according to any one of embodiments 86-89, wherein R²Is H and R³Is methyl substituted by methoxy.

Embodiment 92. Compounds according to any one of embodiments 86-91, wherein R⁴Is hydrogen.

Embodiment 93. Compounds according to any one of embodiments 86-91, wherein R⁴Is an acyl group.

Embodiment 94. the compound of embodiment 86, wherein the compound of formula (2) is

Embodiment 95. the compound of embodiment 86, wherein the compound of formula (2) is

Embodiment 96. the compound of embodiment 86, wherein the compound of formula (2) is

Embodiment 97 the compound of embodiment 86, wherein the compound of formula (2) is

Embodiment 98. compounds of the formula:

or a pharmaceutically acceptable salt thereof, wherein R¹Is heteroaryl or urea, each of which is independently substituted or unsubstituted.

Embodiment 99. the compound of embodiment 98, wherein R¹Is heteroaryl.

Embodiment 100. the compound of embodiment 98 or 99, wherein R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

Embodiment 101. a compound according to embodiment 98, wherein R¹Is a substituted urea.

Embodiment 102 the compound of embodiment 98, wherein the compound is

Embodiment 103. the compound of embodiment 98, wherein the compound is

Embodiment 104. a compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein R¹Is a substituted or unsubstituted heteroaryl; and R is⁵Is hydroxyl or nucleotide.

Embodiment 105 a compound according to embodiment 104, whereinR¹Is a substituted heteroaryl group.

Embodiment 106. a compound according to embodiment 104 or 105, wherein R¹Is 4-amino-2H-1 lambda ²3, 5-triazin-2-one.

Embodiment 107. the composition of embodiments 104 or 105, wherein R¹Is 2-amino-9 lambda²-purin-6 (1H) -one.

Embodiment 108. the compound of any of embodiments 104 to 107, wherein R⁵Is a hydroxyl group.

Embodiment 109. a compound according to any one of embodiments 104 to 107, wherein R⁵Are nucleotides.

Embodiment 110 the compound of embodiment 104, wherein the nucleotide has the formula:

embodiment 111. the compound of embodiment 104, wherein the compound is

Embodiment 112 the compound of embodiment 104, wherein the compound is

Embodiment 113 a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of embodiments 31-58.

Embodiment 114 a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of embodiments 58a-85 b.

Embodiment 115 a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of embodiments 86-112.

Embodiment 116 the method of any one of embodiments 113-115, wherein the condition is cancer.

Embodiment 117. the method of embodiment 116, wherein the cancer is bladder, breast, colon, kidney, epidermis, liver, lung, esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, gastrointestinal system, or skin cancer.

Claims (28)

1. A composition, comprising:

a) a compound of the formula:

or a pharmaceutically acceptable salt thereof,

wherein the composition comprises at least 95% of a compound of formula (1); and

b) a nucleotide-based compound which is not a compound of formula (1).

2. The composition of claim 1, wherein the nucleotide-based compound is a compound of formula (2):

or a pharmaceutically acceptable salt thereof,

wherein:

R¹is heteroaryl or urea, each of which is independently substituted or unsubstituted;

each R²And R³Independently substituted or unsubstituted alkyl; or hydrogen; and is

R⁴Is hydrogen or acyl, each of which is independently substituted or unsubstitutedAnd (4) substitution.

3. The composition of claim 2, wherein R¹Is a substituted urea.

4. The composition of claim 2, wherein R¹Is heteroaryl.

5. The composition of claim 4, wherein R¹Is 4-amino-2H-1 lambda²3, 5-triazin-2-one.

6. The composition of claim 2, wherein each R is²And R³Is substituted alkyl or hydrogen.

7. The composition of claim 6, wherein R²Is H and R³Is methyl substituted by methoxy.

8. The composition of claim 2, wherein R⁴Is hydrogen.

9. The composition of claim 2, wherein R⁴Is an acyl group.

10. The composition of claim 2, wherein the compound of formula (2) is

11. The composition of claim 2, wherein the compound of formula (2) is

12. The composition of claim 2, wherein the compound of formula (2) is

13. The composition of claim 2, wherein the compound of formula (2) is

14. The composition of claim 2, wherein the nucleotide-based compound is a compound of formula (3):

or a pharmaceutically acceptable salt thereof, wherein R¹Is heteroaryl or urea, each of which is independently substituted or unsubstituted.

15. The composition of claim 14, wherein R¹Is heteroaryl.

16. The composition of claim 15, wherein R¹Is 4-amino-2H-1 lambda²3, 5-triazin-2-one.

17. The composition of claim 14, wherein R¹Is a substituted urea.

18. The composition of claim 14, wherein the compound of formula (3) is

19. The composition of claim 14, wherein the compound of formula (3) is

20. The composition of claim 1, wherein the nucleotide-based compound is a compound of formula (4):

or a pharmaceutically acceptable salt thereof, wherein R¹Is a substituted or unsubstituted heteroaryl; and R is⁵Is hydroxyl or nucleotide.

21. The composition of claim 20, wherein R¹Is a substituted heteroaryl group.

22. The composition of claim 21, wherein R¹Is 4-amino-2H-1 lambda²3, 5-triazin-2-one.

23. The composition of claim 21, wherein R¹Is 2-amino-9 lambda²-purin-6 (1H) -one.

24. The composition of claim 20, wherein R⁵Is a hydroxyl group.

25. The composition of claim 20, wherein R⁵Are nucleotides.

26. The composition of claim 25, wherein the nucleotide has the formula:

27. the composition of claim 20, wherein the compound of formula (4) is

28. The composition of claim 20, wherein the compound of formula (4) is

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